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CD82 suppresses melanoma motile machinery by inhibiting U2AF2-mediated CD44v8-10 generation. (A) Lu1205M cells were transfected with vector, CD82, scrambled shRNA, shU2AF2 or shCD44v8-10 before being stained with rhodamine-phalloidin and paxillin antibody (Geen=paxillin, Red=F-actin). Bar=10μm. The average number (B) and size (μm2) (C) of paxillin-containing focal adhesions in transfected Lu1205M were quantified using ImageJ software. 12 cells were analyzed per condition in each experiment. Data were expressed as mean±SEM of three independent experiments. **p < 0.01 compared with control. (D) Lu1205M cells were transfected with vector, CD82, scrambled shRNA, shU2AF2 or shCD44v8-10 before being lysed and subjected to Western blotting analysis of phosphorylation of FAK and Src and GTP-coupling of RhoA. Total FAK, Src, and RhoA were used as loading controls. | nihms-757208-f0012 | 2 | 33bcfab6c4f7221267e16cb34c6fb089009c0fd068d058e0b5b54f0c31b522f9 | nihms-757208-f0012.jpg | multiple | multiple panels: images & plots | [
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"Immuno Assays"
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The downward side of the membrane was stained with crystal violet and migrated cells were counted using an inverted microscope. Image magnification: 200X. Values are shown as mean±SEM from 12 fields from each of three independent experiments. *p<0.05. Bar=50 μm. (E-F) Lu1205M migration potentials under flow conditions were assessed with flow migration assays which were carried out for 4 hr at shear stresses of 0.625 and 2 dyn/cm2. The migrated cells were stained and counted. Values are shown as mean±SEM from 12 fields from each of three independent experiments. *p<0.05.', 'hash': '655b732da9e4e58b69d601f6216514c832edaef7ba5805aed7d6f77ad467027a'}, {'image_id': 'nihms-757208-f0001', 'image_file_name': 'nihms-757208-f0001.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0001.jpg', 'caption': 'CD44v8-10 is highly expressed on metastatic melanoma cell lines. (A) The expressions of central variable regions of CD44 and standard (std) CD44 transcript in NHEM, WM35, SK-Mel-25, A375M and Lu1205M were measured by quantitative real-time PCR with respective primer sets using GAPDH as an endogenous control. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35 cells. *p<0.05 compared with WM35. The structure of CD44 pre-mRNA is shown in the insert. (B) Western blotting analysis of the presence of CD44 isoforms on a variety of melanoma cell lines and NHEM with 2C5 antibody. CD44v:150 kD; CD44s: 100 kD. (C) Verification of the status of 150 kD CD44 on melanoma cell lines with CD44v8-10-secific antibody. (D) Run-off analysis of CD44 isoform expression on Lu1205M, A375M and SK-Mel-25. Exon-specific PCRs were performed using 5’ primers for the variant exons (v2-v10) and constant 5’ primer C13, respectively, in combination with 3’ primer C2A. PCR products were resolved in agarose gel. Numbers in the left indicate marker sizes in bp. Representative results are shown from three independent experiments.', 'hash': 'cb1df0cb6a65b17e6231611c4aaf839f7a3eed99191200e6e4b4a4a5c2759af6'}, {'image_id': 'nihms-757208-f0006', 'image_file_name': 'nihms-757208-f0006.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0006.jpg', 'caption': 'U2AF2 promotes exon v8-10 splicing of CD44 pre-mRNA. (A) Western blotting analysis of the presence of U2AF2 on a variety of metastatic melanoma cell lines and NHEM. (B)\nTop panel, Design and sequence of shRNA-resistant U2AF2 construct are shown. Middle panel, U2AF2 expression in Lu1205M transfected with different constructs was assessed by Western blotting. Bottom panel, The amounts of CD44 standard and variable exons (v1-v10) in WM35 or Lu1205M cells which received scrambled shRNA, shU2AF2 or shU2AF2 plus shRNA-resistant U2AF2 were measured with real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-scrambled cells. *p<0.05 compared with Lu1205M-scrambled. #p<0.05 compared with Lu1205M-shU2AF2. (C) shU2AF2 transfection suppressed CD44v8-10 expression and increased CD44s expression as assessed by Western blotting. (D) The amounts of CD44 standard and variable exons (v1-v10) in WM35 or SK-Mel-25 cells which received vector or U2AF2 were measured with quantitative real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-vector cells. *p<0.05 compared with SK-Mel-25-vector. The U2AF2 expression in SK-Mel-25 cells transfected with different constructs was assessed by Western blotting. (E) The effect of U2AF2 overexpression on CD44 isoform switches in SK-Mel-25 cells was measured by western blotting. (F) Fluorescence microscopy analysis of Lu1205M cells expressing pFlare-V8-10 minigene. Lu1205M/pFlare-V8-10 cells were transfected with either scrambled shRNA or shU2AF2. Bar=50 μm. (G-I) Real-time-PCR analysis of V8-10 exon splicing of CD44 from pFlare-V8-10-expressing stable Lu1205M and A375M cell lines which were transfected with scrambled or U2AF2 shRNA. *p<0.05 compared with scrambled. The V8-10 included product is C-V8-10-GFP; the V8-10 skipped product is C-GFP. (J-K) U2AF2 mediates CD44v8-10 splicing by binding to 3’ polypyrimidine tract flanking V8 exon. Sequences of 3’-splicing site in wild type (WT) and mutant minigenes are shown in the upper panel. Exonic and intronic regions are indicated in uppercase and lowercase, respectively. Alternative splicing events were monitored by real-time PCR. (K) The ratio of the amount of exon V8-10 spliced variant (splicing) to that of V8-10 skipped variant (exclusion) is shown among samples. Data is represented as mean±SEM from three independent assays. *p<0.05 compared with WT.', 'hash': '50f5ea8f00adc558ea308ce9d7389e1b4581d40295590ceb67724a073d5efcf8'}, {'image_id': 'nihms-757208-f0010', 'image_file_name': 'nihms-757208-f0010.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0010.jpg', 'caption': 'CD82 suppresses melanoma invasiveness and migration by modulating U2AF2-dependent CD44 isoform switching. (A) SK-Mel-25 cells that were transfected with vector or U2AF2 were cotransfected with shCD44s or shCD44v8-10. (B) SK-Mel-25 cells that were transfected with scrambled or CD82 siRNA were cotransfected with shU2AF2 or shCD44v8-10. (C, E) Lu1205M cells that were transfected with scrambled or U2AF2 shRNA were cotransfected with CD44s or CD44v8-10. (D, F) Lu1205M cells that were transfected with vector or CD82 were cotransfected with U2AF2 or CD44v8-10. (A-D) SK-Mel-25 or Lu1205M cells were tested for static migration abilities in transwell system. HUVEC monolayer was seeded on transwell insert membrane. The downward side of the membrane was stained with crystal violet and migrated cells were counted using an inverted microscope. Image magnification: 200X. Values are shown as mean±SEM from 12 fields from each of three independent experiments. *p<0.05. Bar=50 μm. (E-F) Lu1205M migration potentials under flow conditions were assessed with flow migration assays which were carried out for 4 hr at shear stresses of 0.625 and 2 dyn/cm2. The migrated cells were stained and counted. Values are shown as mean±SEM from 12 fields from each of three independent experiments. *p<0.05.', 'hash': '959e0b889393c148df6fbb895dbf8b8c2adc6f886ffb084efcd1f72c6364694e'}, {'image_id': 'nihms-757208-f0007', 'image_file_name': 'nihms-757208-f0007.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0007.jpg', 'caption': 'CD82 suppresses U2AF2 activity by mediating U2AF2 ubiquitination. (A-B) The effects of CD82 overexpression in Lu1205M and CD82 knockdown in SK-Mel-25 on U2AF2 expression were measured by Western blotting. (C) Ectopic expression of CD82 has no effect on U2AF2 mRNA levels as measured by real-time PCR using GAPDH as an endogenous control. Lu1205M and A375M cells were transfected with 0, 50, 100 and 200 ng CD82 before mRNA was extracted. Data is represented as mean±SEM from three independent assays. N.S., not significant. (D) Ectopic expression of CD82 enhances U2AF2 ubiquitination. Lu1205M and A375M cells were transfected with 0, 50, 100 and 200 ng CD82 and treated with 10 μM MG132 for 6 hr before harvesting. U2AF2 ubiquitination was detected by immunoprecipitation with U2AF2 antibody and immunoblotting with anti-ubiquitin antibody (ubiq). The results of one of three independent experiments are shown.', 'hash': '76fc35f105a6820c6eca069dfe8e88239f6c1ced82d7772a0b741d7c253fc175'}, {'image_id': 'nihms-757208-f0009', 'image_file_name': 'nihms-757208-f0009.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0009.jpg', 'caption': 'CD82 suppresses melanoma invasiveness and migration by modulating U2AF2-dependent CD44 isoform switching. (A) SK-Mel-25 cells that were transfected with vector or U2AF2 were cotransfected with shCD44s or shCD44v8-10. (B) SK-Mel-25 cells that were transfected with scrambled or CD82 siRNA were cotransfected with shU2AF2 or shCD44v8-10. (C, E) Lu1205M cells that were transfected with scrambled or U2AF2 shRNA were cotransfected with CD44s or CD44v8-10. (D, F) Lu1205M cells that were transfected with vector or CD82 were cotransfected with U2AF2 or CD44v8-10. (A-D) SK-Mel-25 or Lu1205M cells were tested for static migration abilities in transwell system. HUVEC monolayer was seeded on transwell insert membrane. The downward side of the membrane was stained with crystal violet and migrated cells were counted using an inverted microscope. Image magnification: 200X. Values are shown as mean±SEM from 12 fields from each of three independent experiments. *p<0.05. Bar=50 μm. (E-F) Lu1205M migration potentials under flow conditions were assessed with flow migration assays which were carried out for 4 hr at shear stresses of 0.625 and 2 dyn/cm2. The migrated cells were stained and counted. Values are shown as mean±SEM from 12 fields from each of three independent experiments. *p<0.05.', 'hash': '7a6c4b277766728c043511828cd92de4239507905e7fbde983e9bf073ac899c2'}, {'image_id': 'nihms-757208-f0011', 'image_file_name': 'nihms-757208-f0011.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0011.jpg', 'caption': 'U2AF2-mediated CD44 isoform switching increases the affinity of tumor CD44 for E-selectin. Binding affinity of melanoma cells for E-selectin was analyzed by the micropipette assay.(A) Schematic representation of the dual micropipette setup. A CD44-expressing tumor cell and an E-selectin-coated RBC were held by 2 opposing micropipettes and allowed to contact for different time durations. Binding events were recorded by analyzing the deflection of RBC membrane. (B) Adhesion frequency of melanoma to E-selectin upon receptor/ligand contact for 5 sec was increased progressively with increasing metastatic potentials. (C) Lu1205M cells were transfected with vector, CD82, scrambled shRNA, shU2AF2 or shCD44v8-10 and allowed to contact with E-selectin coated RBC for 2.5 and 5 sec before binding frequency was measured. Values are shown as mean±SEM from 6 independent experiments. *p<0.05.', 'hash': '3bcb69e2422a1ed3c57fc3de1037966238d8438905b8039e8195ec4543d795fc'}, {'image_id': 'nihms-757208-f0012', 'image_file_name': 'nihms-757208-f0012.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0012.jpg', 'caption': 'CD82 suppresses melanoma motile machinery by inhibiting U2AF2-mediated CD44v8-10 generation. (A) Lu1205M cells were transfected with vector, CD82, scrambled shRNA, shU2AF2 or shCD44v8-10 before being stained with rhodamine-phalloidin and paxillin antibody (Geen=paxillin, Red=F-actin). Bar=10μm. The average number (B) and size (μm2) (C) of paxillin-containing focal adhesions in transfected Lu1205M were quantified using ImageJ software. 12 cells were analyzed per condition in each experiment. Data were expressed as mean±SEM of three independent experiments. **p < 0.01 compared with control. (D) Lu1205M cells were transfected with vector, CD82, scrambled shRNA, shU2AF2 or shCD44v8-10 before being lysed and subjected to Western blotting analysis of phosphorylation of FAK and Src and GTP-coupling of RhoA. Total FAK, Src, and RhoA were used as loading controls.', 'hash': '33bcfab6c4f7221267e16cb34c6fb089009c0fd068d058e0b5b54f0c31b522f9'}, {'image_id': 'nihms-757208-f0003', 'image_file_name': 'nihms-757208-f0003.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0003.jpg', 'caption': 'Regulation of alternative splicing of CD44 pre-mRNA by CD82. (A) After transfection of WM35 or Lu1205M with a plasmid encoding CD82 or vector alone, amounts of CD44 standard and variable exons (v1-v10) in the central viable region were measured with real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-vector cells. *p<0.05 compared with Lu1205M-vector. CD82 transfection efficiency was assessed by Western blotting. (B) The effect of CD82 overexpression on CD44 isoform switches in Lu1205M was measured by Western blotting. (C) The amounts of standard CD44 standard and variable exons (v1-v10) in SK-Mel-25 cells which received scrambled or CD82 siRNA were measured with quantitative real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35 cells. *p<0.05 compared with SK-Mel-25-siCD82. CD82 knockdown efficiency was assessed by Western blotting. (D) The effect of CD82 knockdown on CD44 isoform switches in SK-Mel-25 cells was measured by Western blotting. Representative results are shown from three independent experiments.', 'hash': 'f23de66ca6a36bff145418c6b483c02846ef4616c4bca575091eedf823dca0af'}, {'image_id': 'nihms-757208-f0004', 'image_file_name': 'nihms-757208-f0004.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0004.jpg', 'caption': 'U2AF2 promotes exon v8-10 splicing of CD44 pre-mRNA. (A) Western blotting analysis of the presence of U2AF2 on a variety of metastatic melanoma cell lines and NHEM. (B)\nTop panel, Design and sequence of shRNA-resistant U2AF2 construct are shown. Middle panel, U2AF2 expression in Lu1205M transfected with different constructs was assessed by Western blotting. Bottom panel, The amounts of CD44 standard and variable exons (v1-v10) in WM35 or Lu1205M cells which received scrambled shRNA, shU2AF2 or shU2AF2 plus shRNA-resistant U2AF2 were measured with real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-scrambled cells. *p<0.05 compared with Lu1205M-scrambled. #p<0.05 compared with Lu1205M-shU2AF2. (C) shU2AF2 transfection suppressed CD44v8-10 expression and increased CD44s expression as assessed by Western blotting. (D) The amounts of CD44 standard and variable exons (v1-v10) in WM35 or SK-Mel-25 cells which received vector or U2AF2 were measured with quantitative real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-vector cells. *p<0.05 compared with SK-Mel-25-vector. The U2AF2 expression in SK-Mel-25 cells transfected with different constructs was assessed by Western blotting. (E) The effect of U2AF2 overexpression on CD44 isoform switches in SK-Mel-25 cells was measured by western blotting. (F) Fluorescence microscopy analysis of Lu1205M cells expressing pFlare-V8-10 minigene. Lu1205M/pFlare-V8-10 cells were transfected with either scrambled shRNA or shU2AF2. Bar=50 μm. (G-I) Real-time-PCR analysis of V8-10 exon splicing of CD44 from pFlare-V8-10-expressing stable Lu1205M and A375M cell lines which were transfected with scrambled or U2AF2 shRNA. *p<0.05 compared with scrambled. The V8-10 included product is C-V8-10-GFP; the V8-10 skipped product is C-GFP. (J-K) U2AF2 mediates CD44v8-10 splicing by binding to 3’ polypyrimidine tract flanking V8 exon. Sequences of 3’-splicing site in wild type (WT) and mutant minigenes are shown in the upper panel. Exonic and intronic regions are indicated in uppercase and lowercase, respectively. Alternative splicing events were monitored by real-time PCR. (K) The ratio of the amount of exon V8-10 spliced variant (splicing) to that of V8-10 skipped variant (exclusion) is shown among samples. Data is represented as mean±SEM from three independent assays. *p<0.05 compared with WT.', 'hash': '09fba9fe5235d085681c6b33367da01d9ac7f6bbe6eb8c6e4774abc5da6a70dc'}, {'image_id': 'nihms-757208-f0013', 'image_file_name': 'nihms-757208-f0013.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0013.jpg', 'caption': 'CD82 regulates melanoma experimental metastasis in vivo by suppressing U2AF2-mediated CD44 isoform switching. (A) Quantification of tumor loci in liver 20 days post implantation of vector, CD82, scrambled shRNA, shU2AF2, shCD44s and shCD44v8-10 transfected-Lu1205M cells into nude mice. Ten mice were injected under each condition. The livers in each group were collected and sectioned. 6 sections through the center of the liver were examined under dissecting microscopy for the presence of metastatic tumor loci. Western blotting analysis was conducted to determine the levels of CD44v8-10 and CD44s in lysates from implanted tumors. (B) Quantification of primary tumor’s tumorigenic potentials. Data are mean±SEM. n=10. (C) Representative images of H&E staining of liver metastases of vector, CD82, scrambled shRNA, shU2AF2 and shCD44v8-10-transfected Lu1205M cells in mice. T: metastatic tumor. (D) IHC staining of U2AF2, CD44s and CD44v8-10 in invasive fronts of primary tumor.', 'hash': '907967fc2f82449c64a56deb4fc42a3bcc74cccf21cc72a1a79167fc07594881'}, {'image_id': 'nihms-757208-f0014', 'image_file_name': 'nihms-757208-f0014.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0014.jpg', 'caption': 'Model of inhibition of U2AF2-dependent CD44 alternative splicing by CD82 in metastatic melanoma. Upper panel, in non-metastatic melanoma, expression of CD82 induces ubiquitination and degradation of U2AF2, which leads to exon V8-10 skipping of CD44 pre-mRNA. Lower panel, loss of CD82 expression results in stabilization and binding of U2AF2 to 3’-splicing site of the intron adjacent to exon V8. This process facilitates exon V8-10 splicing, enhances the binding affinity of CD44 to E-selectin and promotes stress fiber formation and Src/FAK/RhoA activation, leading to increased motility and metastatic potential of melanoma.', 'hash': '27b1fa4ae2e7c80a25aa42bc4d43883282aa6afff2a7dfa8071229b0435abee9'}, {'image_id': 'nihms-757208-f0005', 'image_file_name': 'nihms-757208-f0005.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0005.jpg', 'caption': 'U2AF2 promotes exon v8-10 splicing of CD44 pre-mRNA. (A) Western blotting analysis of the presence of U2AF2 on a variety of metastatic melanoma cell lines and NHEM. (B)\nTop panel, Design and sequence of shRNA-resistant U2AF2 construct are shown. Middle panel, U2AF2 expression in Lu1205M transfected with different constructs was assessed by Western blotting. Bottom panel, The amounts of CD44 standard and variable exons (v1-v10) in WM35 or Lu1205M cells which received scrambled shRNA, shU2AF2 or shU2AF2 plus shRNA-resistant U2AF2 were measured with real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-scrambled cells. *p<0.05 compared with Lu1205M-scrambled. #p<0.05 compared with Lu1205M-shU2AF2. (C) shU2AF2 transfection suppressed CD44v8-10 expression and increased CD44s expression as assessed by Western blotting. (D) The amounts of CD44 standard and variable exons (v1-v10) in WM35 or SK-Mel-25 cells which received vector or U2AF2 were measured with quantitative real-time PCR. Values were expressed as fold changes (mean±SEM, n=3) relative to respective levels in WM35-vector cells. *p<0.05 compared with SK-Mel-25-vector. The U2AF2 expression in SK-Mel-25 cells transfected with different constructs was assessed by Western blotting. (E) The effect of U2AF2 overexpression on CD44 isoform switches in SK-Mel-25 cells was measured by western blotting. (F) Fluorescence microscopy analysis of Lu1205M cells expressing pFlare-V8-10 minigene. Lu1205M/pFlare-V8-10 cells were transfected with either scrambled shRNA or shU2AF2. Bar=50 μm. (G-I) Real-time-PCR analysis of V8-10 exon splicing of CD44 from pFlare-V8-10-expressing stable Lu1205M and A375M cell lines which were transfected with scrambled or U2AF2 shRNA. *p<0.05 compared with scrambled. The V8-10 included product is C-V8-10-GFP; the V8-10 skipped product is C-GFP. (J-K) U2AF2 mediates CD44v8-10 splicing by binding to 3’ polypyrimidine tract flanking V8 exon. Sequences of 3’-splicing site in wild type (WT) and mutant minigenes are shown in the upper panel. Exonic and intronic regions are indicated in uppercase and lowercase, respectively. Alternative splicing events were monitored by real-time PCR. (K) The ratio of the amount of exon V8-10 spliced variant (splicing) to that of V8-10 skipped variant (exclusion) is shown among samples. Data is represented as mean±SEM from three independent assays. *p<0.05 compared with WT.', 'hash': 'd53e8474b6c0304cb3f33b06cf2b2f3e7326906ba4095a6dfca516bb042dccf1'}, {'image_id': 'nihms-757208-f0002', 'image_file_name': 'nihms-757208-f0002.jpg', 'image_path': '../data/media_files/PMC5033661/nihms-757208-f0002.jpg', 'caption': 'CD44v8-10 expression is positively correlated with U2AF2 expression and negatively correlated with CD82 level in metastatic melanoma tissues. (A) Analysis of expressions of CD44v8-10 and U2AF2 with IHC in nevi from 6 non-melanoma patients and 12 samples of melanoma in situ and metastatic melanoma from melanoma patients. Protein levels were quantified in FFPE tissue samples. Mann-Whitney U test was used. (B) The correlation between CD82 relative expression levels (log transformed) and CD44v8-10 relative expression levels (log transformed) in 70 FFPE melanoma tissue samples from patients with clinical TNM stage 0-IV. Pearson’s R test was used. R- and p-values are calculated as indicated. (C) Association between CD44v8-10 and U2AF2 expression and survival of melanoma patients (n=70). The melanoma patients were stratified according to CD44v8-10 and U2AF2 expression levels into high and low groups. Patients expressing high CD44v8-10 and U2AF2 levels displayed significantly shorter survival (log-rank test).', 'hash': '06f1f2e1faf78ef27337bccae2ab4da580d2c927b0ab73e4426c1a0c202d04cb'}] | {'nihms-757208-f0001': ['CD44 variant isoforms are upregulated in cancer stem cell-like cells and advanced cancer, playing roles in tumor adhesion, hematogenous metastasis, drug resistance and tumorigenesis 6, 20-23. To identify the CD44 isoform present on malignant melanoma, real-time PCR was employed to measure variable exon selections in Lu1205M, A375M 2, SK-Mel-25 and WM35 cells with different metastatic potentials (Table 1) as well as Normal Human Epidermal Melanocytes (NHEMs). Lu1205M cells were derived from Lu1205 cells 4 and underwent extensive aggressiveness selection. Therefore, Lu1205M cells exhibited high malignant and metastatic properties with elevated potentials for colonizing lungs and livers (Table 1). Real-time PCR revealed that SK-Mel-25 cells expressed appreciable amounts of CD44s but low levels of variable exons, while exons 12-14 (v8-v10) were selectively expressed on A375M and Lu1205M cells (<xref ref-type="fig" rid="nihms-757208-f0001">Fig. 1A</xref>). The performance of each primer set was evaluated with CD44v2-10-expressing keratinocyte HaCaT cells. All primer sets efficiently amplified target exons in HaCaT mRNA (). The performance of each primer set was evaluated with CD44v2-10-expressing keratinocyte HaCaT cells. All primer sets efficiently amplified target exons in HaCaT mRNA (Supplementary Fig.1A). PCR results were validated with Western blotting (2C5 antibody) which demonstrated that NHEM and WM35 don’t express any CD44 molecules, SK-Mel-25 only expresses CD44s yielding an intense 100 kD band, A375M expresses the 100 kD CD44s and a 150 kD protein, and high levels of the 150 kD protein were also detectable on Lu1205M cells (<xref ref-type="fig" rid="nihms-757208-f0001">Fig. 1B</xref>). PCR results motivated us to surmise that the 150 kD protein is CD44v8-10. To verify the status of the protein, we generated a CD44v8-10 epitope-specific antibody (). PCR results motivated us to surmise that the 150 kD protein is CD44v8-10. To verify the status of the protein, we generated a CD44v8-10 epitope-specific antibody (Supplementary Fig. 2A-B). This antibody specifically recognized the 150 kD protein without any cross-reactivity with other proteins on Lu1205M and A375M cells, suggesting that the protein is CD44v8-10 (<xref ref-type="fig" rid="nihms-757208-f0001">Fig. 1C</xref>). The lack of reactivity of VFF-18 towards any epitopes in SK-Mel-25, A375M and Lu1205M samples implies that melanoma cells don’t express detectable amounts of CD44v6 protein (). The lack of reactivity of VFF-18 towards any epitopes in SK-Mel-25, A375M and Lu1205M samples implies that melanoma cells don’t express detectable amounts of CD44v6 protein (Supplementary Fig. 3A). A more precise determination of the exon composition of variant isoforms was achieved by the run-off analysis which verified the continuous alignment of v8, v9 and v10 exons up to CD44v8-10 (729 bp) in the transcripts from Lu1205M and A375M cells, while the predominant isoform on SK-Mel-25 cells was CD44s (268 bp) (<xref ref-type="fig" rid="nihms-757208-f0001">Fig. 1D</xref>). As a positive control, exon-specific primers used in the run-off analysis detected CD44v2-10, CD44v6-10 and CD44v8-10 which are the major CD44v isoforms being expressed by HaCaT (). As a positive control, exon-specific primers used in the run-off analysis detected CD44v2-10, CD44v6-10 and CD44v8-10 which are the major CD44v isoforms being expressed by HaCaT (Supplementary Fig. 1B).'], 'nihms-757208-f0002': ['To evaluate the clinical implication of molecular alteration of CD44, we analyzed melanoma patient tissue samples (Table 2). We included melanoma in situ which is designated as TNM stage 0 (TisN0M0) into our analysis. In 70 melanoma patients, CD44v8-10 staining was not correlated with age and gender, while it was significantly correlated with tumor differentiation status and TNM stages. Strong CD44v8-10 expression was found in 20% and 40% cases with stage I and II, while 73% and 80% of stage III and IV melanoma had strong CD44v8-10 expression. CD44v8-10 staining intensity increased in primary melanoma compared with dysplastic nevi and further rose in metastatic melanomas (p<0.001) (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2A</xref>). Nevi, primary melanoma and metastatic melanoma showed a weak expression of CD44v6 (). Nevi, primary melanoma and metastatic melanoma showed a weak expression of CD44v6 (Supplementary Fig. 3B). No significant difference in staining intensity of CD44v6 was found in nevi compared to primary and metastatic melanoma (p>0.05). In analysis of Kaplan-Meier survival curves, we found that elevated CD44v8-10 was significantly correlated with a poorer overall survival of all melanoma patients (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2C</xref>). These data suggest that elevated CD44v8-10 may have pathological roles in melanoma metastasis.). These data suggest that elevated CD44v8-10 may have pathological roles in melanoma metastasis.', 'Then, we determined the association between CD82 and CD44v8-10 expression in clinical samples. CD82 level tended to drop as the TNM staging advanced and high abundance of CD82 correlated with a better relapse-free survival (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2B</xref>). In addition, CD82 expression was negatively correlated with levels of CD44v8-10 as detected by CD44v8-10 antibody and commercial CD44v7/8, CD44v9 and CD44v10 antibodies (). In addition, CD82 expression was negatively correlated with levels of CD44v8-10 as detected by CD44v8-10 antibody and commercial CD44v7/8, CD44v9 and CD44v10 antibodies (p<0.001, R=0.76) (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2B</xref> and and Supplementary Fig. 5).', 'As with CD44v8-10, enhanced expression of U2AF2 was observed during clinical melanoma progression (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2A</xref>). U2AF2 level was drastically increased in primary melanoma compared with dysplastic nevi (P <0.001) and further upregulated in metastatic melanoma (P<0.001). In addition, decreased U2AF2 staining was strongly associated with a better patient survival (). U2AF2 level was drastically increased in primary melanoma compared with dysplastic nevi (P <0.001) and further upregulated in metastatic melanoma (P<0.001). In addition, decreased U2AF2 staining was strongly associated with a better patient survival (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2C</xref>). The 5-year survival rate dropped from 75% in patients with low U2AF2 expression to 48% in those with positive U2AF2 expression. There was a significant negative correlation between CD82 and U2AF2 levels in melanoma samples (). The 5-year survival rate dropped from 75% in patients with low U2AF2 expression to 48% in those with positive U2AF2 expression. There was a significant negative correlation between CD82 and U2AF2 levels in melanoma samples (<xref ref-type="fig" rid="nihms-757208-f0002">Fig. 2B</xref>). Altogether, these results suggest that in metastatic melanoma, downregulation of CD82 expression elevated U2AF2 expression, which increased the ratio of CD44v8-10 to CD44s.). Altogether, these results suggest that in metastatic melanoma, downregulation of CD82 expression elevated U2AF2 expression, which increased the ratio of CD44v8-10 to CD44s.'], 'nihms-757208-f0003': ['Previously, it was reported that endothelial CD82 inhibited the cell surface presence of CD4419. Furthermore, downregulation of CD82 is common in advanced human cancer24. Therefore, we examined the effect of modulation of CD82 expression on CD44 isoform expression in melanoma. Compared with WM35, Lu1205M expresses low levels of CD82 (<xref ref-type="fig" rid="nihms-757208-f0003">Fig. 3A</xref>). When CD82 was overexpressed in Lu1205M, CD44v8-10 was reduced and CD44s upregulated at both mRNA and protein levels (). When CD82 was overexpressed in Lu1205M, CD44v8-10 was reduced and CD44s upregulated at both mRNA and protein levels (<xref ref-type="fig" rid="nihms-757208-f0003">Fig. 3A-B</xref> and and Supplementary Fig. 4A-B). Conversely, knockdown of endogenous CD82 with siRNA in SK-Mel-25 resulted in a remarkable increase in CD44v8-10 and a concomitant reduction of CD44s expression (<xref ref-type="fig" rid="nihms-757208-f0003">Fig. 3C-D</xref>). These results suggest that CD82 functions as an important regulator to suppress the isoform switch from CD44s to CD44v.). These results suggest that CD82 functions as an important regulator to suppress the isoform switch from CD44s to CD44v.'], 'nihms-757208-f0004': ['To investigate whether CD82 suppresses CD44v8-10 expression through regulation of splicing, we silenced a set of splicing factors, Tra2β, SRp20, ESRP1, YB-1, SRm160, Sam68 and U2AF2, which have been shown to mediate CD44v splicing 10, 12, 13, 25, 26. Only knockdown of U2AF2 dramatically reduced CD44v8-10 expression (Supplementary Fig. 6). Of five cell lines, U2AF2 expression was associated with metastatic potentials with the highest level found on Lu1205M (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4A</xref>). U2AF2 knockdown with shRNA significantly increased CD44s and attenuated CD44v8-10 expressions (). U2AF2 knockdown with shRNA significantly increased CD44s and attenuated CD44v8-10 expressions (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4B-C</xref>, , Supplementary Fig. 4C-D). Then, we generated a shRNA-resistant U2AF2 construct which contains three mutated nucleotides in shRNA seeding region without changing amino acid sequence (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4B</xref>\n\nupper panel)27. Overexpression of shRNA-resistant U2AF2 in U2AF2-silenced Lu1205M rescued CD44v8-10 expression, suggesting that U2AF2 is the splicing factor mediating CD44s/CD44v isoform switch (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4B</xref>\n\nmiddle and lower panels). To further evaluate the role of U2AF2 in CD44 alternative splicing, U2AF2 construct was transfected into SK-Mel-25 cells which expressed low levels of this splicing factor. Overexpression of U2AF2 triggered CD44v8-10 expression at both mRNA and protein levels (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4D-E</xref>).).', 'To gain more insight into the molecular basis for the function of U2AF2 in regulating CD44 splicing, we utilized a minigene with pFlare-RFP/GFP reporter system28 where start condon for GFP is split by inserting V8-10 exon and its flanking introns. This insertion results in GFP expression when V8-10 exon is skipped and RFP synthesis when it is included. Our results showed that RFP was dominantly expressed, suggesting that CD44v8-10 is spliced in control Lu1205M (<xref ref-type="fig" rid="nihms-757208-f0004">Fig.4F</xref>). In contrast, knockdown of U2AF2 in Lu1205M attenuated RFP signal and increased GFP expression. Knockdown of U2AF2 in Lu1205M and A375M promoted exon skipping and significantly reduced the ratio of V8-10 exon splicing/exclusion (). In contrast, knockdown of U2AF2 in Lu1205M attenuated RFP signal and increased GFP expression. Knockdown of U2AF2 in Lu1205M and A375M promoted exon skipping and significantly reduced the ratio of V8-10 exon splicing/exclusion (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4G-I</xref>). U2AF2 can bind to weak polypyrimidine tract in 3’-splicing site to facilitate exon splicing). U2AF2 can bind to weak polypyrimidine tract in 3’-splicing site to facilitate exon splicing26. To find whether 3’-splicing site of V8-10 contains U2AF2 response element, we introduced mutations in 3’ polypyrimidine tract. Point mutations from CT to AA at −4 and −5 positions sharply comprised the stimulative effect of U2AF2 on V8-10 splicing (<xref ref-type="fig" rid="nihms-757208-f0004">Fig. 4J-K</xref>). These results imply that by targeting 3’ polypyrimidine tract, U2AF2 enforces CD44 v8-10 splicing in melanoma.). These results imply that by targeting 3’ polypyrimidine tract, U2AF2 enforces CD44 v8-10 splicing in melanoma.'], 'nihms-757208-f0007': ['To demonstrate that U2AF2 is under control of CD82 to mediate CD44 splicing, U2AF2 expression was examined in Lu1205M following CD82 overexpression. Ectopic expression of CD82 in Lu1205M diminished U2AF2 expression (<xref ref-type="fig" rid="nihms-757208-f0007">Fig. 5A</xref>). On the other hand, knockdown of CD82 by siRNA in SK-Mel-25 resulted in an increased expression of U2AF2 (). On the other hand, knockdown of CD82 by siRNA in SK-Mel-25 resulted in an increased expression of U2AF2 (<xref ref-type="fig" rid="nihms-757208-f0007">Fig. 5B</xref>). Surprisingly, although CD82 knockdown in Lu1205M and A375M induced a dramatic change of protein levels of U2AF2, mRNA levels did not correlate with this change (). Surprisingly, although CD82 knockdown in Lu1205M and A375M induced a dramatic change of protein levels of U2AF2, mRNA levels did not correlate with this change (<xref ref-type="fig" rid="nihms-757208-f0007">Fig. 5C</xref>). Therefore, CD82 may modulate U2AF2 activities at posttranslational level. Accordingly, we tested the hypothesis that CD82 reduces U2AF2 protein stability by inducing its ubiquitination. Our results showed that CD82 increased U2AF2 ubiquitination in a dose-dependent manner in both Lu1205M and A375M (). Therefore, CD82 may modulate U2AF2 activities at posttranslational level. Accordingly, we tested the hypothesis that CD82 reduces U2AF2 protein stability by inducing its ubiquitination. Our results showed that CD82 increased U2AF2 ubiquitination in a dose-dependent manner in both Lu1205M and A375M (<xref ref-type="fig" rid="nihms-757208-f0007">Fig. 5D</xref>).).'], 'nihms-757208-f0008': ['Since CD44v isoform expression in cancer may contribute to tumor adhesion, migration and metastasis, we next assessed the roles of CD82 in CD44v8-10-dependent cell motility6, 29. Overexpression of U2AF2 significantly enhanced SK-Mel-25 migration (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6A</xref>). However, the pro-migratory effect of U2AF2 can be attenuated by CD44v8-10 shRNA but not by CD44s shRNA. When endogenous CD82 in SK-MEL-25 were inhibited, cell migration was enhanced by 5-fold (). However, the pro-migratory effect of U2AF2 can be attenuated by CD44v8-10 shRNA but not by CD44s shRNA. When endogenous CD82 in SK-MEL-25 were inhibited, cell migration was enhanced by 5-fold (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6B</xref>). Knockdown of U2AF2 or CD44v8-10 markedly reduced the migration potentials of SK-Mel-25/siCD82 cells. Either knockdown of U2AF2 or overexpression of CD82 in Lu1205M suppressed Lu1205M motility (). Knockdown of U2AF2 or CD44v8-10 markedly reduced the migration potentials of SK-Mel-25/siCD82 cells. Either knockdown of U2AF2 or overexpression of CD82 in Lu1205M suppressed Lu1205M motility (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6C and D</xref>). Restoration of CD44v8-10 but not CD44s rescued the suppressive effect of shU2AF2 on melanoma migration (). Restoration of CD44v8-10 but not CD44s rescued the suppressive effect of shU2AF2 on melanoma migration (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6C</xref>). Similarly, migratory potentials of Lu1205M/CD82 were increased by reconstitution of U2AF2 or CD44v8-10 (). Similarly, migratory potentials of Lu1205M/CD82 were increased by reconstitution of U2AF2 or CD44v8-10 (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6D</xref>). A lack of involvement of CD44v6 in melanoma migration was evident as knockdown of CD44v6 did not affect Lu1205M migration (). A lack of involvement of CD44v6 in melanoma migration was evident as knockdown of CD44v6 did not affect Lu1205M migration (Supplementary Fig. 3C). These results suggest that CD82 suppresses melanoma motility through regulating U2AF2 expression and CD44s/CD44v8-10 isoform switching.', 'Flow regulated cancer migration plays important roles in tumor metastasis. To evaluate the roles of CD82-regulated CD44 splicing on melanoma migration under hydrodynamic conditions, we utilized a flow migration assay1, 30. At a shear stress of 0.625 dyn/cm2 but not 2 dyn/cm2, knockdown of endogenous U2AF2 or overexpression of CD82 reduced the transendothelial migration of Lu1205M (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6E and F</xref>). Conversely, restoration of CD44v8-10 but not CD44s abrogated the U2AF2 knockdown-dependent reduction in melanoma extravasation (). Conversely, restoration of CD44v8-10 but not CD44s abrogated the U2AF2 knockdown-dependent reduction in melanoma extravasation (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6E</xref>). The suppressive effect of CD82 overexpression on melanoma migration in flow was rescued by transfection of U2AF2 or CD44v8-10 (). The suppressive effect of CD82 overexpression on melanoma migration in flow was rescued by transfection of U2AF2 or CD44v8-10 (<xref ref-type="fig" rid="nihms-757208-f0008">Fig. 6F</xref>). Therefore, CD44v8-10 may contribute to melanoma migration under low shear conditions.). Therefore, CD44v8-10 may contribute to melanoma migration under low shear conditions.'], 'nihms-757208-f0011': ['Cell tethering mediated by reversible interactions between selectins and sialyl-LewisX-decorated receptors on tumor surface is the first step for tumor metastasis31, 32. To determine the effect of CD44 isoform switching on tumor adhesion, we assessed binding frequency between E-selectin and melanoma with a micropipette aspiration assay (<xref ref-type="fig" rid="nihms-757208-f0011">Fig. 7A</xref>). Upon 5-sec contacts, CD44s-expressing SK-Mel-25 adhered more frequently to E-selectin compared with WM35 or NHEM (). Upon 5-sec contacts, CD44s-expressing SK-Mel-25 adhered more frequently to E-selectin compared with WM35 or NHEM (p<0.05) (<xref ref-type="fig" rid="nihms-757208-f0011">Fig. 7B</xref>). CD44v8-10-expressing A375M or Lu1205M had higher affinity for E-selectin than SK-Mel-25 (). CD44v8-10-expressing A375M or Lu1205M had higher affinity for E-selectin than SK-Mel-25 (p<0.05). Overexpression of CD82 or knockdown of U2AF2 or CD44v8-10 sharply diminished the adhesion probability of Lu1205M (<xref ref-type="fig" rid="nihms-757208-f0011">Fig. 7C</xref>). Interestingly, the difference of adhesion probability among different cell lines was not observed for short contact duration (2.5 sec) (). Interestingly, the difference of adhesion probability among different cell lines was not observed for short contact duration (2.5 sec) (<xref ref-type="fig" rid="nihms-757208-f0011">Fig. 7B-C</xref>).).'], 'nihms-757208-f0012': ['In migrating Lu1205M, robust stress fiber structures formed with thick actin filaments traversing the cell bodies (<xref ref-type="fig" rid="nihms-757208-f0012">Fig. 8A</xref>). In sharp contrast, ectopic expression of CD82 disrupted the stress fiber structure with only thin filaments visible in the cell peripheries. Likewise, silencing U2AF2 or CD44v8-10 abolished actin polymerization. By staining paxillin, a scaffold adaptor protein localized in focal adhesion, it was demonstrated that control cells had bright punctate focal adhesion associated with the ends of thick stress fibers. But overexpression of CD82 or knockdown of U2AF2 or CD44v8-10 reduced the size and number of focal adhesions where paxillin spots disengaged with actin (). In sharp contrast, ectopic expression of CD82 disrupted the stress fiber structure with only thin filaments visible in the cell peripheries. Likewise, silencing U2AF2 or CD44v8-10 abolished actin polymerization. By staining paxillin, a scaffold adaptor protein localized in focal adhesion, it was demonstrated that control cells had bright punctate focal adhesion associated with the ends of thick stress fibers. But overexpression of CD82 or knockdown of U2AF2 or CD44v8-10 reduced the size and number of focal adhesions where paxillin spots disengaged with actin (<xref ref-type="fig" rid="nihms-757208-f0012">Fig. 8A-C</xref>). Then, the activation status of migratory machinery, including focal adhesion kinase (FAK), Src, and RhoA, was assessed. CD82 overexpression or knockdown of U2AF2 or CD44v8-10 suppressed the phosphorylation of FAK and Src and GTP-coupling of RhoA without affecting total FAK, Src and RhoA levels (). Then, the activation status of migratory machinery, including focal adhesion kinase (FAK), Src, and RhoA, was assessed. CD82 overexpression or knockdown of U2AF2 or CD44v8-10 suppressed the phosphorylation of FAK and Src and GTP-coupling of RhoA without affecting total FAK, Src and RhoA levels (<xref ref-type="fig" rid="nihms-757208-f0012">Fig. 8D</xref>).).'], 'nihms-757208-f0013': ['To assess whether modulation of CD82, U2AF2, CD44s and CD44v8-10 expression could affect melanoma metastasis in vivo, we used two metastatic models: xenograft liver metastasis and experimental lung metastasis. Compared with control groups, xenograft tumors derived from CD82-overexpressing, U2AF2-slienced and CD44v8-10-silenced Lu1205M metastasized less efficiently to the liver 20 days after subcutaneous implantation (<xref ref-type="fig" rid="nihms-757208-f0013">Fig. 9A</xref>). CD82 overexpression, U2AF2 knockdown or CD44v8-10 knockdown reduced the number of metastatic lesions by 80%. On the contrary, CD44s knockdown did not significantly reduce Lu1205M liver metastasis compared with control. To determine whether melanoma tumorigenesis was affected, size of primary tumors was measured on alternative days up to 19.5 days. Overexpression of CD82 or knockdown of U2AF2 or CD44v8-10 did not alter the rate at which Lu1205M tumors grew in comparison to control (). CD82 overexpression, U2AF2 knockdown or CD44v8-10 knockdown reduced the number of metastatic lesions by 80%. On the contrary, CD44s knockdown did not significantly reduce Lu1205M liver metastasis compared with control. To determine whether melanoma tumorigenesis was affected, size of primary tumors was measured on alternative days up to 19.5 days. Overexpression of CD82 or knockdown of U2AF2 or CD44v8-10 did not alter the rate at which Lu1205M tumors grew in comparison to control (<xref ref-type="fig" rid="nihms-757208-f0013">Fig. 9B</xref>). Thus, CD82-regulated CD44v8-10 splicing had no effect on melanoma tumorigenesis. H&E staining verified tumor nodule quantification results for involvement of CD82, U2AF2 and CD44v8-10 in melanoma liver metastasis (). Thus, CD82-regulated CD44v8-10 splicing had no effect on melanoma tumorigenesis. H&E staining verified tumor nodule quantification results for involvement of CD82, U2AF2 and CD44v8-10 in melanoma liver metastasis (<xref ref-type="fig" rid="nihms-757208-f0013">Fig. 9C</xref>). In tumor migrating fronts, CD44v8-10 was localized in cell membrane, while U2AF2 exhibited a nucleus and cytoplasma staining pattern (). In tumor migrating fronts, CD44v8-10 was localized in cell membrane, while U2AF2 exhibited a nucleus and cytoplasma staining pattern (<xref ref-type="fig" rid="nihms-757208-f0013">Fig. 9D</xref>). CD44s was absent from tumor migrating fronts. CD82 overexpression or U2AF2 knockdown reduced the robust staining for CD44v8-10 and U2AF2, whereas CD44s levels were increased (). CD44s was absent from tumor migrating fronts. CD82 overexpression or U2AF2 knockdown reduced the robust staining for CD44v8-10 and U2AF2, whereas CD44s levels were increased (<xref ref-type="fig" rid="nihms-757208-f0013">Fig. 9D</xref>). As for experimental lung metastasis, Lu1205M-LUC/CD82 cells migrated less efficiently than control cells (). As for experimental lung metastasis, Lu1205M-LUC/CD82 cells migrated less efficiently than control cells (Supplementary Fig. 7A and B). Further, overexpression of U2AF2 or CD44v8-10 rescued tumor lung metastasis. Notably, inoculation of Lu1205M-LUC co-transfected with CD82 and CD44v8-10 resulted in even more lung seedings than Lu1205M-LUC/vector melanoma.'], 'nihms-757208-f0014': ['Our study revealed that CD44v8-10 is not only a prognostic marker but also a functional receptor for melanoma metastasis. In agreement with earlier studies, our results showed that elevated level of CD44v8-10 is associated with tumor metastatic potentials11. It was reported that CD44v8-10 splicing promotes tumorigenesis by driving stem cell-like properties25, 33. In the current study, our findings provided novel evidence for the role of CD82 in regulating pre-mRNA splicing in melanoma and proposed a mechanism by which CD82 suppresses melanoma metastasis by modulating U2AF2-dependent CD44v8-10 splicing (<xref ref-type="fig" rid="nihms-757208-f0014">Fig. 10</xref>). This hypothesis is supported by: 1) overexpression of CD82 in highly metastatic melanoma reduces the ratio of CD44v8-10 to CD44s; 2) CD82 regulates U2AF2 protein stability by facilitating its ubiquitination; 3) overexpression of CD82 in melanoma impedes tumor migration under static and flow conditions, which can be rescued by restoration of U2AF2 or CD44v8-10 expression; 4) the suppressive effect of CD82 on tumor migration is dependent on altered affinity of CD44 for selectin, inactivation of Src/FAK/RhoA, and disassembly of stress fibers and focal adhesions.). This hypothesis is supported by: 1) overexpression of CD82 in highly metastatic melanoma reduces the ratio of CD44v8-10 to CD44s; 2) CD82 regulates U2AF2 protein stability by facilitating its ubiquitination; 3) overexpression of CD82 in melanoma impedes tumor migration under static and flow conditions, which can be rescued by restoration of U2AF2 or CD44v8-10 expression; 4) the suppressive effect of CD82 on tumor migration is dependent on altered affinity of CD44 for selectin, inactivation of Src/FAK/RhoA, and disassembly of stress fibers and focal adhesions.']} | CD82 suppresses CD44 alternative splicing-dependent melanoma metastasis by mediating U2AF2 ubiquitination and degradation | [
"CD82",
"U2AF2",
"CD44v",
"alternative splicing"
] | Oncogene | 1474527600 | Melanoma is one of the most lethal forms of skin cancer because of its early metastatic spread. The variant form of CD44 (CD44v), a cell surface glycoprotein, is highly expressed on metastatic melanoma. The mechanisms of regulation of CD44 alternative splicing in melanoma and its pathogenic contributions are so far poorly understood. Here, we investigated the expression level of CD44 in a large set of melanocytic lesions at different stages. We found that the expression of CD44v8-10 and a splicing factor, U2AF2, is significantly increased during melanoma progression, whereas CD82/KAI1, a tetraspanin family of tumor suppressor, is reduced in metastatic melanoma. CD44v8-10 and U2AF2 expression levels, which are negatively correlated with CD82 levels, are markedly elevated in primary melanoma compared with dysplastic nevi and further increased in metastatic melanoma. We also showed that patients with higher CD44v8-10 and U2AF2 expression levels tended to have shorter survival. By using both in vivo and in vitro assays, we demonstrated that CD82 inhibits the production of CD44v8-10 on melanoma. Mechanistically, U2AF2 is a downstream target of CD82 and in malignant melanoma facilitates CD44v8-10 alternative splicing. U2AF2-mediated CD44 isoform switch is required for melanoma migration in vitro and lung and liver metastasis in vivo. Notably, overexpression of CD82 suppresses U2AF2 activity by inducing U2AF2 ubiquitination. In addition, our data suggested that enhancement of melanoma migration by U2AF2-dependent CD44v8-10 splicing is mediated by Src/focal adhesion kinase/RhoA activation and formation of stress fibers, as well as CD44-E-selectin binding reinforcement. These findings uncovered a hitherto unappreciated function of CD82 in severing the linkage between U2AF2-mediated CD44 alternative splicing and cancer aggressiveness, with potential prognostic and therapeutic implications in melanoma. | [
"Alternative Splicing",
"Animals",
"Cell Line, Tumor",
"Gene Expression Regulation, Neoplastic",
"Humans",
"Hyaluronan Receptors",
"Kangai-1 Protein",
"Melanocytes",
"Melanoma",
"Mice",
"Neoplasm Metastasis",
"Phosphorylation",
"Prognosis",
"Protein Isoforms",
"Proteolysis",
"Splicing Factor U2AF",
"Ubiquitination",
"Xenograft Model Antitumor Assays"
] | other | PMC5033661 | null | 52 | [
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] | Oncogene. 2016 Sep 22; 35(38):5056-5069 | NO-CC CODE |
|
Tra-IR700 mediated photoimmunotherapy for HER2 expressing and non expressing co-cultured cells(a) Induction of target specific photoimmunotherapy (PIT) lead to HER2 expressing cell specific necrotic cell death. Scale bar, 50 μm. (See also Supplementary Video 2) (b) HER2 specific cell death was confirmed with fluorescence microscopy with LIVE/DEAD Green staining. Scale bar, 100 μm. (c) Flow cytometric analysis for detecting HER2-specific cell death induced by Tra-IR700 (TraIR) mediated PIT. Upper left quadrant: Tra-IR700 positive, live cells; upper right quadrant; Tra-IR700 positive, dead cells; lower left quadrant: Tra-IR700 negative, live cells; lower right quadrant: Tra-IR700 negative, dead cells (n = 3). DIC: differential interference contrast. | nihms282847f4 | 2 | ddd6c2aa9fd6607002b9b56bb8f9a6dc64a24a25e2eb2d2ea64bafa56600d1d1 | nihms282847f4.jpg | multiple | multiple panels: images & plots | [
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] | [
"fluorescence microscopy",
"bar plot",
"immunoblot"
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800,
467
] | [{'image_id': 'nihms282847f1', 'image_file_name': 'nihms282847f1.jpg', 'image_path': '../data/media_files/PMC3233641/nihms282847f1.jpg', 'caption': '(a) A schema for explaining selective cancer therapy with photoimmunotherapy (PIT) in the context of other physical cancer therapies employing electro-magnetic wave irradiation. Although other physical cancer therapies induce different types of damages in the normal tissue, PIT dedicatedly damages cancer cells without damaging normal cells or tissues. (b) A schema for explaining photo-physical, chemical and biological basis of PIT. Humanized antibodies are employed as a delivary vehicle from the biology and medicine points of view because of its highest binding specificity, greatest in vivo target delivery, low immunogenecity among the clinically applicable targeting reagents. A hydrophilic phtalocyanine is employed as a activatable cytotoxic “Nano-dynamite” reagent from the chemistry points of view because of its great absorption of near infrared light of 700nm and strong cytotoxicity induced only when associating with the cell membrane. Near infrared light of 700nm is employed as an initiator for activating cytotoxicity from the physics points of view because of its high energy among non-harmful non-ionizing photons and great in vivo tissue penetration.', 'hash': '9c1f4aed4f591fbd884e4bb6398d1459654b61f5bccae90c7edcfea9d7305bda'}, {'image_id': 'nihms282847f2', 'image_file_name': 'nihms282847f2.jpg', 'image_path': '../data/media_files/PMC3233641/nihms282847f2.jpg', 'caption': "Target specific cell death in response to Tra-IR700 mediated photoimmunotherapy for 3T3/HER2 cells(a) Different subcellular localization of Tra-IR700 (TraIR). Scale bar, 30 μm. (b) Lysosomal localization of Tra-IR700 6 h after incubation. Scale bar, 50 μm. (c) Microscopic observation of before and after Tra-IR700 mediated photoimmunotherapy (PIT). Scale bar, 50 μm. (d) Irradiation dose dependent and target specific cell death in response to Tra-IR700 mediated PIT. Data are means ± s.e.m. (n = at least 4, *** P < 0.001 vs. non treatment control, Student's t test). (e) Long term growth inhibition in response to Tra-IR700 mediated PIT. Data are means ± s.e.m. (n = 3, ** P < 0.01 vs. non treatment control, Student's t test). (f) Microscopic observation of growth inhibition in response to Tra-IR700 mediated PIT. Scale bar, 100 μm (g) Internalization of Tra-IR700 was not required for phototoxic cell death. Data are means ± s.e.m. (n = 3). (h) Target specific membrane binding of Tra-IR700 only induced phototoxic cell death. Data are means ± s.e.m. (n = 3). (i) HER2 negatively expressing A431 cells did not show phototoxic effects with Tra-IR700 mediated PIT (n = 3). (j) Sodium azide (NaN3) concentration dependent inhibition of phototoxic cell death induced by Tra-IR700 mediated PIT. Data are means ± s.e.m. (n = 3, *** P < 0.001, ** P < 0.01 vs. 2.0 J cm-2 PIT treatment without NaN3 control, Student's t test). DIC: differential interference contrast. PanIR: Pan-IR700.", 'hash': '463edea1d1e46df3456aa04e7302776f69c170c89bb18e15a045edae78ec2959'}, {'image_id': 'nihms282847f5', 'image_file_name': 'nihms282847f5.jpg', 'image_path': '../data/media_files/PMC3233641/nihms282847f5.jpg', 'caption': "Pan-IR700 mediated photoimmunotherapy for HER1 expressing tumors in vivo(a) HER1 positive A431 tumor (left dorsum) was selectively visualized as early as 1 d after Pan-IR700 injection (50 μg). HER1 negative 3T3/HER2 tumor (right dorsum) did not show detectable fluorescence (n = 5 mice). (b) Fluorescence intensity of IR700 in A431 tumors over time at two different dose of Pan-IR700. Data are means ± s.e.m. (n = 4 each mice). (c) Tumor to background ratio of IR700 fluorescence intensity in A431 tumors over time at two different dose of Pan-IR700. Data are means ± s.e.m. (n = 4 each mice). (d) Biodistribution of Pan-IR700. A431 tumors (both sides of dorsum) were selectively visualized with IR700 fluorescence as early as 1 d after Pan-IR700 injection (300 μg). Right side of the tumor was irradiated with near infrared (NIR) light on day 1, while left side of the tumor was covered with black tape. Tumor shrinkage was confirmed on day 7. Dashed line: irradiated tumor, solid line: non-irradiated tumor. (e) Target specific tumor growth inhibition by Pan-IR700 mediated photoimmunotherapy (PIT) for A431 tumors. PIT was performed on day 1 after Pan-IR700 injection (day 5 after tumor inoculation). Data are means ± s.e.m. (at least n = 12 mice in each group, *** P < 0.001 vs. other control groups, Kruskal–Wallis test with post-test). (f) Kaplan-Meier survival curve analysis of Pan-IR700 mediated PIT for A431 tumors (at least n = 12 mice in each group, *** P < 0.001 vs. other control groups, log-rank test with Bonferroni's correction for multiplicity). (g) Histological observation of treated and non-treated A431 tumors (n = 5 mice, hematoxylin and eosin staining). Scale bar, 100 μm. Pan: panitumumab.", 'hash': '67071c057febeac3e76e7048877fd03bdfb997cbd24bda31beda40a5ad9d7962'}, {'image_id': 'nihms282847f4', 'image_file_name': 'nihms282847f4.jpg', 'image_path': '../data/media_files/PMC3233641/nihms282847f4.jpg', 'caption': 'Tra-IR700 mediated photoimmunotherapy for HER2 expressing and non expressing co-cultured cells(a) Induction of target specific photoimmunotherapy (PIT) lead to HER2 expressing cell specific necrotic cell death. Scale bar, 50 μm. (See also Supplementary Video 2) (b) HER2 specific cell death was confirmed with fluorescence microscopy with LIVE/DEAD Green staining. Scale bar, 100 μm. (c) Flow cytometric analysis for detecting HER2-specific cell death induced by Tra-IR700 (TraIR) mediated PIT. Upper left quadrant: Tra-IR700 positive, live cells; upper right quadrant; Tra-IR700 positive, dead cells; lower left quadrant: Tra-IR700 negative, live cells; lower right quadrant: Tra-IR700 negative, dead cells (n = 3). DIC: differential interference contrast.', 'hash': 'ddd6c2aa9fd6607002b9b56bb8f9a6dc64a24a25e2eb2d2ea64bafa56600d1d1'}, {'image_id': 'nihms282847f3', 'image_file_name': 'nihms282847f3.jpg', 'image_path': '../data/media_files/PMC3233641/nihms282847f3.jpg', 'caption': "Target specific cell death in response to Pan-IR700 mediated photoimmunotherapy for EGFR expressing A431 cells(a) Microscopic observation of before and after Pan-IR700 mediated photoimmunotherapy (PIT). Scale bar, 50 μm. (b) Irradiation dose dependent and target specific cell death in response to Pan-IR700 (PanIR) mediated PIT. Data are means ± s.e.m. (n = at least 4, *** P < 0.001 vs. non treatment control, Student's t test). (c) EGFR expressing cell specific necrotic cell death was induced by Pan-IR700 mediated PIT. Scale bar, 50 μm. DIC: differential interference contrast.", 'hash': 'e46655a8831f68ec8ef98cbd9e1af59e817a05a7ec4e0dfe6b2ae8202238831f'}] | {'nihms282847f1': ['Conventional photodynamic therapy (PDT), which combines a photosensitizing agent with the physical energy of non-ionizing light to kill cells, has been less commonly employed for cancer therapy because the current non-targeted photosensitizers are also taken up in normal tissues, thus, causing serious side effects, although the excitation light itself is harmless in the near infrared (NIR) range (<xref rid="nihms282847f1" ref-type="fig">Fig. 1</xref>). Were it feasible to design a highly targeted photosensitizer, toxicity could be greatly reduced. Although the targeted photosensitizer may distribute throughout the body, it will only be active where intense light is applied, reducing the likelihood of off-target effects. Most existing photosensitizers are poorly selective small molecules which bind not only to cancer cells but also to normal cells including the skin and other epithelial surfaces resulting in unwanted phototoxicity. These agents are generally hydrophobic, therefore, permeate into cells and produce reactive oxygen species intracellularly, leading to cell death. Thus, target specific delivery of conventional photosensitizers is theoretically difficult because, after reaching the cell, the agent must still be internalized into organelles, such as mitochondria, to be most effective. Various combinations of conventional photosensitizers and MAbs have been tested to improve selectivity with limited success especially when measured by ). Were it feasible to design a highly targeted photosensitizer, toxicity could be greatly reduced. Although the targeted photosensitizer may distribute throughout the body, it will only be active where intense light is applied, reducing the likelihood of off-target effects. Most existing photosensitizers are poorly selective small molecules which bind not only to cancer cells but also to normal cells including the skin and other epithelial surfaces resulting in unwanted phototoxicity. These agents are generally hydrophobic, therefore, permeate into cells and produce reactive oxygen species intracellularly, leading to cell death. Thus, target specific delivery of conventional photosensitizers is theoretically difficult because, after reaching the cell, the agent must still be internalized into organelles, such as mitochondria, to be most effective. Various combinations of conventional photosensitizers and MAbs have been tested to improve selectivity with limited success especially when measured by in vivo therapeutic effects5-11. There are several reasons for unsuccessful outcomes; 1. Conventional photosensitizers have low extinction coefficients that requires conjugation of large numbers of photosensitizers to a single antibody molecule thus, potentially decreasing binding affinity. 2. Conventional photosensitizers are mostly hydrophobic leading to difficulties in conjugating photosensitizers to antibodies without compromising the immunoreactivity and in vivo target accumulation. 3. Conventional photosensitizers generally absorb light in the visible range reducing tissue penetration.', 'The NIR excitation light wavelength (peaking at 689 nm) allows penetration of at least several centimeters into tissues16. By using fiber-coupled laser diodes with diffuser tips, NIR light can be delivered within several centimeters of otherwise inaccessible tumors located deep to the body surface. Using such fibers, PDT has been used to treat brain tumors and peritoneal metastasis of ovarian cancer27. In addition to treating solid cancers it may be possible to target circulating tumor cells since they could be excited when they traverse superficial vessels. Although no toxicity was observed in our experiments, clinical translation will require formal toxicity studies. In addition, free IR700 and catabolized IR700, are readily excreted into urine within 1 hour without accumulation in any specific organ (Supplementary Fig. 8). The other component of PIT, light irradiation with NIR at 690 nm wavelength is unlikely to be toxic except at thermal doses. Theoretically, there should be no limitations on the cumulative irradiation dose of NIR light, unlike ionizing radiation such as x-ray or gamma-ray (<xref rid="nihms282847f1" ref-type="fig">Fig. 1</xref>). Therefore, repeated PIT might be possible for long term management of some cancer patients. In reality, repeated PIT with 3 different regimens (3 or 4 fractionated NIR irradiations at a single dose of MAb-IR700 and 4 cycles of PIT every 2 weeks after multiple doses of antibody) controlled tumor re-growth, resulting in tumor free survival of more than 4 months.). Therefore, repeated PIT might be possible for long term management of some cancer patients. In reality, repeated PIT with 3 different regimens (3 or 4 fractionated NIR irradiations at a single dose of MAb-IR700 and 4 cycles of PIT every 2 weeks after multiple doses of antibody) controlled tumor re-growth, resulting in tumor free survival of more than 4 months.', 'In conclusion, we have developed a target specific PIT based on MAb-IR700 conjugate (<xref rid="nihms282847f1" ref-type="fig">Fig. 1</xref>). The photosensitizer, IR700, is excited in the NIR range leading to deeper tissue penetration resulting in successful eradication of subcutaneously xenografted tumors after only a single dose of external NIR light irradiation. Targeted phototoxicity seems to be primarily dependent on binding of the MAb-IR700 to the cell membrane and to a lesser extent on internalization and ROS formation. The ability to covalently conjugate any number of different antibodies to IR700 means that this may be a highly flexible theranostic platform. The fluorescence induced by the conjugate can be used to non-invasively guide both PIT and monitor the results of therapy. Thus, the MAb-IR700 conjugate is a promising therapeutic and diagnostic agent for the treatment of cancer.). The photosensitizer, IR700, is excited in the NIR range leading to deeper tissue penetration resulting in successful eradication of subcutaneously xenografted tumors after only a single dose of external NIR light irradiation. Targeted phototoxicity seems to be primarily dependent on binding of the MAb-IR700 to the cell membrane and to a lesser extent on internalization and ROS formation. The ability to covalently conjugate any number of different antibodies to IR700 means that this may be a highly flexible theranostic platform. The fluorescence induced by the conjugate can be used to non-invasively guide both PIT and monitor the results of therapy. Thus, the MAb-IR700 conjugate is a promising therapeutic and diagnostic agent for the treatment of cancer.'], 'nihms282847f2': ['Fluorescence microscopy was performed to visualize the cellular binding location of the conjugates. Consistent with previous studies, IR700 fluorescence was detected mainly on the cell surface of 3T3/HER2 cells, which express HER2 protein, after 1 h incubation at 4 °C with Tra-IR700, whereas the fluorescence was also detected inside the cells after 6 h incubation at 37 °C with Tra-IR700, indicating gradual internalization12 (<xref rid="nihms282847f2" ref-type="fig">Fig. 2a</xref>). Co-staining with LysoTracker-Green revealed co-localization of IR700 with the endolysosomal compartment (). Co-staining with LysoTracker-Green revealed co-localization of IR700 with the endolysosomal compartment (<xref rid="nihms282847f2" ref-type="fig">Fig. 2b</xref>). After 1 h and 6 h of incubation with Tra-IR700, excitation light (fluorescence microscope; power density of 2.2 mW cm). After 1 h and 6 h of incubation with Tra-IR700, excitation light (fluorescence microscope; power density of 2.2 mW cm-2) induced fluorescence as well as cellular swelling, bleb formation, and rupture of vesicles representing necrotic cell death (<xref rid="nihms282847f2" ref-type="fig">Fig. 2c</xref> and and Supplementary Video 1a, b). Necrotic cell death was also observed using HER1 positive A431 cells incubated with Pan-IR700 which were exposed to excitation light as described above (<xref rid="nihms282847f3" ref-type="fig">Fig. 3a</xref>).).', 'To further explore the nature of the phototoxicity of Tra-IR700 on 3T3/HER2 cells we used the LIVE/DEAD assay to analyze acute phototoxicity and trypan blue dye exclusion assay to analyze the effect on proliferation. As cell death was induced rapidly upon irradiation (Supplementary Video 1a, b), the LIVE/DEAD assay, which can detect the cells with damaged membranes was performed within 1 h after the treatment. The percentage of cell death in target cells vs. untreated control cells was significantly influenced by excitation light dose (<xref rid="nihms282847f2" ref-type="fig">Fig. 2d</xref>). In addition, there was no significant cytotoxicity associated with exposure to Tra-IR700 without excitation light or with light exposure without Tra-IR700. Similar results were obtained for A431 cells with Pan-IR700 (). In addition, there was no significant cytotoxicity associated with exposure to Tra-IR700 without excitation light or with light exposure without Tra-IR700. Similar results were obtained for A431 cells with Pan-IR700 (<xref rid="nihms282847f3" ref-type="fig">Fig. 3b</xref>), however, panitumumab itself had a noticeable treatment effect against A431 cells due to down regulation and signal inhibition of HER1), however, panitumumab itself had a noticeable treatment effect against A431 cells due to down regulation and signal inhibition of HER113. In addition, parental 3T3 cells served as HER2 receptor-negative controls, therefore, we chose 3T3/HER2 cells with Tra-IR700 for further in vitro study. Proliferation assay revealed that long term growth inhibition was confirmed only when cells were treated with Tra-IR700 and exposed to light (<xref rid="nihms282847f2" ref-type="fig">Fig. 2e, f</xref> and and Supplementary Fig. 2a).', 'There was no significant difference in phototoxicity between 1 h and 6 h incubation with Tra-IR700 (<xref rid="nihms282847f2" ref-type="fig">Fig. 2g</xref>), indicating that membrane binding of Tra-IR700 was sufficient to induce cell death. When Tra-IR700 was localized to the endolysosomal compartment (), indicating that membrane binding of Tra-IR700 was sufficient to induce cell death. When Tra-IR700 was localized to the endolysosomal compartment (<xref rid="nihms282847f2" ref-type="fig">Fig. 2b</xref>), it also induced rupture of the vesicle with cellular swelling and bleb formation after irradiation (), it also induced rupture of the vesicle with cellular swelling and bleb formation after irradiation (Supplementary Video 1a). However, this did not appear to be a major cause of cell death, since cell death could be observed without endolysosomal localization of Tra-IR700 within 1 h of incubation at 4 °C (Supplementary Video 1b). Interestingly, failure to wash the cells prior to irradiation did not influence the phototoxic effect indicating that cellular membrane binding was important to the phototoxic effects of the conjugate, not merely the presence of the conjugate. Further, the IR700 dye alone (200 nM; equivalent IR700 concentration of Tra-IR700 conjugates) did not incorporate into the cells or induce phototoxcity in cells (<xref rid="nihms282847f2" ref-type="fig">Fig. 2h</xref> and and Supplementary Fig. 2b). Additionally, phototoxcity was dose-dependently blocked by the excess unconjugated trastuzumab (Supplementary Fig. 2c, d). Furthermore, Tra-IR700 did not induce therapeutic effect to A431 cells (<xref rid="nihms282847f2" ref-type="fig">Fig. 2i</xref>). These results confirm that cell death is dependent on specific membrane binding of Tra-IR700.). These results confirm that cell death is dependent on specific membrane binding of Tra-IR700.', 'Reactive oxygen species (ROS) have been implicated in the cell death associated with conventional PDT. To clarify the role of photon-induced redox reactions (e.g. singlet oxygen (1O2)) in producing phototoxicity with Tra-IR700, a redox quencher, sodium azide (NaN3)14, was added to the medium when cells were irradiated. The percentage of cell death was partially decreased in the presence of sodium azide, in a dose dependent manner (<xref rid="nihms282847f2" ref-type="fig">Fig. 2j</xref>).).', 'The selectivity of the MAb-IR700 is derived from its activation after binding to the cell membrane of target cells; unbound conjugate does not contribute to phototoxicity (<xref rid="nihms282847f2" ref-type="fig">Fig. 2a, g, h</xref>). Short term viability assays, as well as long term proliferation assays, demonstrated that the conjugate was capable of inducing specific cell death (). Short term viability assays, as well as long term proliferation assays, demonstrated that the conjugate was capable of inducing specific cell death (<xref rid="nihms282847f2" ref-type="fig">Fig. 2d–f</xref>). When co-cultures of receptor-positive and -negative cells were treated, only the receptor-positive cells were killed despite the presence of unbound MAb-IR700 in the culture medium (). When co-cultures of receptor-positive and -negative cells were treated, only the receptor-positive cells were killed despite the presence of unbound MAb-IR700 in the culture medium (<xref rid="nihms282847f4" ref-type="fig">Fig. 4a–c</xref>; see also ; see also Supplementary Video 2). This selective cell killing minimizes damage to normal cells.', 'Although the mechanism of phototoxicity with MAb-IR700 is not completely clear, the agent must be bound to the cellular membrane to be active. Treatment with sodium azide, a well known redox and singlet oxygen scavenger, only partially reduced the phototoxicity but did not totally eliminate the effectiveness of the conjugate (<xref rid="nihms282847f2" ref-type="fig">Fig. 2j</xref>). This indicates that ROS generation is a minor part of the phototoxic effect. The observation that phototoxicity was induced after incubation with MAb-IR700 after only 1 h at 4 °C, indicates that internalization of the conjugate is not required for activity (). This indicates that ROS generation is a minor part of the phototoxic effect. The observation that phototoxicity was induced after incubation with MAb-IR700 after only 1 h at 4 °C, indicates that internalization of the conjugate is not required for activity (<xref rid="nihms282847f2" ref-type="fig">Fig. 2g</xref>, and , and Supplementary Video 1b). This differs from the current generation of PDT agents that require intracellular localization to be effective. Video microscopy demonstrated rapid visible damage to the membrane and lysosomes after exposure to light, following incubation for more than 6 h at 37 °C, when the MAb-conjugate was internalized (Supplementary Video 1a). Previous studies have indicated that intracellular uptake of MAb-photosensitizer conjugates is responsible for inducing phototoxicity6-10. While this new MAb-IR700 conjugate does not require internalization for phototoxic cell death, cell surface antigen binding is required (<xref rid="nihms282847f2" ref-type="fig">Fig. 2g, h</xref>). For instance, the rupture of endolysosome occurred within a second of light exposure (). For instance, the rupture of endolysosome occurred within a second of light exposure (Supplementary video 1a). Cell death induced by singlet oxygen generally induces a slower apoptotic cell death (Supplementary Fig. 5a, b)19,20. Since cell membrane damage was so quickly induced even at 4 °C by this method, it is hypothesized that cell death is caused by the rapid expansion of locally heated water with relatively minor effects due to singlet oxygen effects (<xref rid="nihms282847f2" ref-type="fig">Fig.2g, j</xref>, and , and Supplementary video 1a, b)21.'], 'nihms282847f4': ['To confirm that the phototoxicity was target specific, we next treated 3T3/HER2 and Balb/3T3/DsRed, which is a parental HER2 negative Balb/3T3 transfected with DsRed fluorescent protein. Tra-IR700 was distributed in a HER2 specific manner while DsRed expressing Balb/3T3 cells did not show phototoxicity upon irradiation (<xref rid="nihms282847f4" ref-type="fig">Fig. 4a</xref> and and Supplementary Video 2). In addition, LIVE/DEAD Green staining demonstrated HER2 specific induction of cell death as determined by multi-color fluorescence microscopy (<xref rid="nihms282847f4" ref-type="fig">Fig. 4b</xref>) and flowcytometry analysis () and flowcytometry analysis (<xref rid="nihms282847f4" ref-type="fig">Fig. 4c</xref>). Target-specific phototoxicity was also confirmed with Pan-IR700 mediated PIT in A431 cells and Balb/3T3/DsRed (HER1 negative) co-cultured cells (). Target-specific phototoxicity was also confirmed with Pan-IR700 mediated PIT in A431 cells and Balb/3T3/DsRed (HER1 negative) co-cultured cells (<xref rid="nihms282847f3" ref-type="fig">Fig. 3c</xref>). Overall, Tra-IR700 and Pan-IR700 showed identical therapeutic effects to HER2 positive (3T3/HER2) and HER1 positive (A431) cells, respectively, except that unconjugated panitumumab showed noticeable growth inhibition but unconjugated trastzumab did not reduce growth with the dose used.). Overall, Tra-IR700 and Pan-IR700 showed identical therapeutic effects to HER2 positive (3T3/HER2) and HER1 positive (A431) cells, respectively, except that unconjugated panitumumab showed noticeable growth inhibition but unconjugated trastzumab did not reduce growth with the dose used.'], 'nihms282847f5': ['To examine the conjugate distribution in vivo, we prepared a xenograft tumor model bearing A431 (HER1 positive) and 3T3/HER2 (HER1 negative) tumors in each dorsum of a mouse. A431 tumors were visualized with IR700 fluorescence 1 d after intravenous injection of Pan-IR700 (50 μg) (<xref rid="nihms282847f5" ref-type="fig">Fig. 5a</xref>). The fluorescence intensity of Pan-IR700 in a A431 tumor decreased gradually over days, while tumor to background ratios (TBRs) increased (). The fluorescence intensity of Pan-IR700 in a A431 tumor decreased gradually over days, while tumor to background ratios (TBRs) increased (<xref rid="nihms282847f5" ref-type="fig">Fig. 5b, c</xref>). The fluorescence intensity of the 3T3/HER2 tumor was the same as that of background (non-tumor lesions). When 300 μg of Pan-IR700 was administered intravenously, fluorescence intensity of the A431 tumor was more than 3 times higher than 50 μg injection at 1 d after injection, however, TBR was lower because of high background signal (). The fluorescence intensity of the 3T3/HER2 tumor was the same as that of background (non-tumor lesions). When 300 μg of Pan-IR700 was administered intravenously, fluorescence intensity of the A431 tumor was more than 3 times higher than 50 μg injection at 1 d after injection, however, TBR was lower because of high background signal (<xref rid="nihms282847f5" ref-type="fig">Fig. 5b, c</xref>). As less antitumor activity was found in mice receiving 50 μg (vs. 300 μg) of Pan-IR700 injection following irradiation (). As less antitumor activity was found in mice receiving 50 μg (vs. 300 μg) of Pan-IR700 injection following irradiation (Supplementary Fig. 3), therefore, we chose the higher injection dose for the treatment study. Biodistribution of Tra-IR700 was determined with IR700 fluorescence because tissue levels of radioactivity and fluorescence might be different due to their different excretion routes and catabolism when using dual-labeled radiolabeled-Pan-IR70015. There was no other specific localization of IR700 except for bladder accumulation on day 1 probably due to excretion of catabolized and unbound dye (<xref rid="nihms282847f5" ref-type="fig">Fig. 5d</xref>, and , and Supplementary Fig. 4a).', 'A431 tumors were treated with a single dose of light at 1 d after Pan-IR700 administration. The efficacy of Pan-IR700 mediated PIT was studied in 8 groups of A431 tumor bearing mice (at least n = 12 mice in each group). All treated tumors were less than 500 mm3 as larger tumors were associated with side effects (i.e. subcutaneous bleeding, tumor bleeding or weakened state) requiring euthanasia in accordance with our institution\'s animal care and use guidelines. Tumor volume was significantly reduced in A431 tumors treated with Pan-IR700 PIT compared with non treatment control mice (<xref rid="nihms282847f5" ref-type="fig">Fig. 5e</xref>), and survival was significantly prolonged in Pan-IR700-PIT treated mice (), and survival was significantly prolonged in Pan-IR700-PIT treated mice (<xref rid="nihms282847f5" ref-type="fig">Fig. 5f</xref>). No significant therapeutic effect was observed in other control groups of mice. Similar results were obtained in 3T3/HER2 tumors treated with Tra-IR700 PIT (). No significant therapeutic effect was observed in other control groups of mice. Similar results were obtained in 3T3/HER2 tumors treated with Tra-IR700 PIT (Supplementary Fig. 4b). Pathological analysis revealed that only scant viable A431 tumor cells were present after Pan-IR700 mediated PIT and massive granulation with inflammatory change was observed in the tumor nodule (<xref rid="nihms282847f5" ref-type="fig">Fig. 5g</xref>). It was also observed that tissue edema developed superficially. To assess the acute phase toxicity of Pan-IR700, we repeatedly administrated 300 μg of Pan-IR700 intravenously twice a week for 4 w, but there were no adverse effects observed up to 8 w (). It was also observed that tissue edema developed superficially. To assess the acute phase toxicity of Pan-IR700, we repeatedly administrated 300 μg of Pan-IR700 intravenously twice a week for 4 w, but there were no adverse effects observed up to 8 w (n = 4) compared with the control group.', 'Another desirable feature of PIT using fluorescent MAb-IR700 conjugate is that it permitted the detection of targeted tissue. Theoretically, this would allow specific lesions to be identified with PIT rather than irradiating the entire field. Doses required for diagnosis (50 μg), however, were significantly lower than those required for therapy (300 μg) Improved intratumoral distribution of antibody occurred with the therapeutic dose (Supplementary Fig. 6)22,23. Because both bound and unbound agent fluoresces, there is relatively high background signal at therapeutic doses (<xref rid="nihms282847f5" ref-type="fig">Fig. 5a–c</xref>). Nevertheless, after PIT, the fluorescence of the treated tumors decreased and eventually disappeared, suggesting a potential means of monitoring the treatment (). Nevertheless, after PIT, the fluorescence of the treated tumors decreased and eventually disappeared, suggesting a potential means of monitoring the treatment (<xref rid="nihms282847f5" ref-type="fig">Fig. 5d</xref>).).', 'One difficulty in interpreting the in vivo results of this study was that trastuzumab, but not panitumumab, demonstrated minor therapeutic effects by itself without the application of light. Since panitumumab is an IgG2 and trastuzumab is an IgG1, this was likely mediated by ADCC and CDC effects even though non-saturating doses were administered (<xref rid="nihms282847f5" ref-type="fig">Fig. 5e, f</xref> and and Supplementary Fig. 4b)24. Based on the similarity of the phototoxicity induced with three different MAbs against several different cells expressing various numbers of respective target molecules and considering the potentially additive benefits from immunotherapy (Supplementary Fig. 7), we believe that this method may be generally applicable to other MAbs25,26.']} | In Vivo Cancer Cell-Selective Near Infrared Photoimmunotherapy Targeting Specific Membrane Molecules | null | Nat Med | 1320562800 | Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. Here we show that proinflammatory T cells inhibit the ability of exogenously added bone marrow mesenchymal stem cells (BMMSCs) to mediate bone repair. This inhibition is due to interferon γ (IFN-γ)-induced downregulation of the runt-related transcription factor 2 (Runx-2) pathway and enhancement of tumor necrosis factor α (TNF-α) signaling in the stem cells. We also found that, through inhibition of nuclear factor κB (NF-κB), TNF-α converts the signaling of the IFN-γ-activated, nonapoptotic form of TNF receptor superfamily member 6 (Fas) in BMMSCs to a caspase 3- and caspase 8-associated proapoptotic cascade, resulting in the apoptosis of these cells. Conversely, reduction of IFN-γ and TNF-α concentrations by systemic infusion of Foxp3(+) regulatory T cells, or by local administration of aspirin, markedly improved BMMSC-based bone regeneration and calvarial defect repair in C57BL/6 mice. These data collectively show a previously unrecognized role of recipient T cells in BMMSC-based tissue engineering. | [
"Animals",
"Apoptosis",
"Aspirin",
"Bone Marrow Cells",
"Bone Regeneration",
"Caspase 3",
"Caspase 8",
"Cell Survival",
"Female",
"Forkhead Transcription Factors",
"Interferon-gamma",
"Mesenchymal Stem Cells",
"Mice",
"Mice, Inbred C57BL",
"Mice, Transgenic",
"NF-kappa B",
"Osteogenesis",
"Signal Transduction",
"T-Lymphocytes, Regulatory",
"Tissue Engineering",
"Tumor Necrosis Factor-alpha"
] | other | PMC3233641 | null | 40 | [
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] | Nat Med. 2011 Nov 6; 17(12):1685-1691 | NO-CC CODE |
|
Effect of Nudel on MT organization during spindle assembly induced by AurA-beads and RanGTP. (a and b) Progression of spindle morphogenesis induced by AurA-beads. (a) Early asters formed in the first 1’–3’ had astral MTs (white arrowhead) attached to the bead surface. Late asters formed at ~6’ had an increasing density of MT astral arrays (white arrowhead) attached to a clear bright MT core (outlined by a white dashed circle). MT balls, which appeared after 9’, had an expanding bright MT core (outlined by white dashed circles) surrounding the beads and with time the astral MT arrays (white arrowheads) became fewer and shorter than those seen in early and late asters. Most structures were MT balls and spindles at 13’–15’. (b) Quantification of different MT structures at the indicated time point. (c) Bacterially-expressed and purified GST or GST-tagged N-terminal half (amino acids 1–201) of Nudel was analyzed by SDS-PAGE followed by Coomassie blue staining. (d) Effects of excess purified GST-N-Nudel on MT morphogenesis during spindle assembly induced by AurA-beads. GST-N-Nudel or GST was added at ~50-fold molar excess of endogenous Nudel (50–100 nM). Whereas MT balls formed in the presence of GST had dense MT cores (white arrow) associated with a thin and short array of MTs (white arrowhead), asters formed in the presence of GST-N-Nudel had long MT arrays (yellow arrowheads) attached to a MT core that was not as densely packed with MTs as the controls (compare the structures pointed to by white and yellow arrows). (e) Effects of Nudel depletion on MT morphogenesis during spindle assembly induced by AurA-beads. Depletion of Nudel from Xenopus egg extracts resulted in a severe block of MT ball and spindle assembly, which were rescued by addition of purified 6His-Nudel. Examples of spindles, MT balls, and MT asters are shown. White arrows and arrowheads point to the dense MT core and short MT arrays, respectively, of MT balls found in mock depleted or Nudel depleted and rescued extracts. Yellow arrows and arrowheads point to the less densely packed MT core and long MT arrays, respectively, of MT asters found in Nudel-depleted egg extracts. Rhodamine-labeled tubulin was used for MT visualization. Images were acquired using a confocal microscope (Leica SP5). Scale, magnetic beads (2.8 µm in diameter). Error bars, standard deviation from >3 independent experiments. | nihms100611f3 | 2 | cdc1930ac4d5ba542cbd0dd5423b9a2c89341b4f94733b58e3e65bc49ef759dd | nihms100611f3.jpg | multiple | multiple panels: images & plots | [
"Microscopy",
"Plots and Charts",
"Immuno Assays"
] | [
"fluorescence microscopy",
"bar plot",
"immunoblot"
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1050,
743
] | [{'image_id': 'nihms100611f1', 'image_file_name': 'nihms100611f1.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f1.jpg', 'caption': 'Interaction of Nudel and dynein with LB3. (a) Nudel antibodies specifically recognized purified full length 6His-Nudel and Nudel in Xenopus egg extracts by Western blotting. (b) Antibodies to Nudel immunoprecipitated (IP) LB3 in egg extracts as judged by Western blotting (WB) analysis. (c) Antibodies to LB3 immunoprecipitated dynein, as detected by 70.1 antibody, in egg extracts. (d) Antibodies to LB3 or Nudel did not pull-down tubulin from the egg extracts. Immunoprecipitations were carried out in the presence of nocodazole to depolymerize MTs. Equivalent of 0.1 and 10 µl of extracts were loaded for input and for immunoprecipitations, respectively, except for the lanes that show LB3 pull-down LB3 itself where only the equivalent of 0.2 µl of egg extract was loaded. (e) Bacterially expressed and purified 6His-tagged full length, N-, or C-terminus of Nudel were analyzed by SDS-PAGE followed by Coommasie blue staining. (f) Beads coupled with purified GST-full length LB3 pulled down purified 6His-Nudel. GST-coupled beads or empty beads served as controls. Comparable amount of GST and GST-LB3 were loaded on the beads as judged by Coomassie Blue staining. 0.033% of the input Nudel and 33% of the precipitate were used for Western blotting (WB). (g) Beads coupled with purified GST-LB3-Rod pulled down more 6His-Nudel than beads coupled with GST-LB3T. GST-bound beads or empty beads served as controls. 0.016% of the input Nudel and 16% of the precipitate were loaded for Western blotting. (h) Beads coupled with purified GST-LB3-coil2, but not GST-LB3-coil1, pulled down 6His-Nudel. 0.033% of the input Nudel and 16% of the precipitate were loaded for Western blotting. (i) Beads coupled with either purified GST-LB3-Rod or purified GST-LB3-coil2, but not GST-LB3-coil1, pulled down purified 6His-C-Nudel. (j) Beads coupled with purified GST-LB3-Rod, GST-LB3-coil1, or GST-LB3-coil2 all failed to pull down 6His-N-Nudel. In both (i) and (j), 0.041% of the input Nudel and 33% of the precipitate were loaded for Western blotting.', 'hash': '0f855ac54efca383a63daf90e42247324b029ac7b87eba2b89a57aa44145ce19'}, {'image_id': 'nihms100611f6', 'image_file_name': 'nihms100611f6.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f6.jpg', 'caption': 'Nudel promotes assembly of the spindle matrix and MT organization. (a) Effects of Nudel depletion on assembly of LB3-containing matrices. In the absence of Nudel, most beads were not associated with LB3-containing matrices, while only a few beads were associated with LB3-containing matrices that were smaller in size compared to controls. Addition of purified 6His-Nudel rescued formation of LB3 matrices (quantifications on the right were carried out blindly). (b) Effects of LB3 depletion on formation of matrices containing Nudel and dynein. Depletion of LB3 still allowed the formation of Nudel and dynein-containing matrices (quantifications on the right were carried out blindly). (c) Effects of LB3 depletion on MT structures induced by AurA-beads. Shown are representative spindles and MT balls formed in mock- and LB3-depleted egg extracts. Rhodamine tubulin and Nudel antibodies were used to visualize MT structures. (d and e) Quantification of the diameter of MT balls and the length of spindles at 15’ of reactions. 40–150 structures assembled were measured in each category. All images were acquired on a Leica SP5 confocal microscope with laser lines used indicated in parentheses. Scale, magnetic beads (2.8 µm in diameter). Error bars, standard deviation from >3 independent experiments.', 'hash': '03fae7a59ab0324efec1c4f9ba6439aa14a9e2d811efaebfab14ae98bbb2fab2'}, {'image_id': 'nihms100611f7', 'image_file_name': 'nihms100611f7.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f7.jpg', 'caption': 'Effects of LB3 depletion on MT structures induced by sperm chromatin. (a) Assembly of MT asters in the first 15’ was not affected by LB3 depletion. (b) Depletion of LB3 disrupted the organization of polarized MT arrays toward the sperm chromatin after 15’ (compare white and yellow arrows and arrowheads). (c) Depletion of LB3 resulted in spindle defects at 45’–120’, including multipolar spindles, spindles with splayed poles, and spindles with few MTs. White arrows point to the defective spindle poles. DAPI (blue) stained the sperm DNA. All quantifications were carried out blindly. All images were acquired on a Leica SP5 confocal microscope. Scale bars, 10 µm. Error bars, standard deviation from >3 independent experiments.', 'hash': '6d6dbd2a8d8c034a41a1f5888fccf8d18226b857eddfb84f1c7aef39087e4476'}, {'image_id': 'nihms100611f3', 'image_file_name': 'nihms100611f3.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f3.jpg', 'caption': 'Effect of Nudel on MT organization during spindle assembly induced by AurA-beads and RanGTP. (a and b) Progression of spindle morphogenesis induced by AurA-beads. (a) Early asters formed in the first 1’–3’ had astral MTs (white arrowhead) attached to the bead surface. Late asters formed at ~6’ had an increasing density of MT astral arrays (white arrowhead) attached to a clear bright MT core (outlined by a white dashed circle). MT balls, which appeared after 9’, had an expanding bright MT core (outlined by white dashed circles) surrounding the beads and with time the astral MT arrays (white arrowheads) became fewer and shorter than those seen in early and late asters. Most structures were MT balls and spindles at 13’–15’. (b) Quantification of different MT structures at the indicated time point. (c) Bacterially-expressed and purified GST or GST-tagged N-terminal half (amino acids 1–201) of Nudel was analyzed by SDS-PAGE followed by Coomassie blue staining. (d) Effects of excess purified GST-N-Nudel on MT morphogenesis during spindle assembly induced by AurA-beads. GST-N-Nudel or GST was added at ~50-fold molar excess of endogenous Nudel (50–100 nM). Whereas MT balls formed in the presence of GST had dense MT cores (white arrow) associated with a thin and short array of MTs (white arrowhead), asters formed in the presence of GST-N-Nudel had long MT arrays (yellow arrowheads) attached to a MT core that was not as densely packed with MTs as the controls (compare the structures pointed to by white and yellow arrows). (e) Effects of Nudel depletion on MT morphogenesis during spindle assembly induced by AurA-beads. Depletion of Nudel from Xenopus egg extracts resulted in a severe block of MT ball and spindle assembly, which were rescued by addition of purified 6His-Nudel. Examples of spindles, MT balls, and MT asters are shown. White arrows and arrowheads point to the dense MT core and short MT arrays, respectively, of MT balls found in mock depleted or Nudel depleted and rescued extracts. Yellow arrows and arrowheads point to the less densely packed MT core and long MT arrays, respectively, of MT asters found in Nudel-depleted egg extracts. Rhodamine-labeled tubulin was used for MT visualization. Images were acquired using a confocal microscope (Leica SP5). Scale, magnetic beads (2.8 µm in diameter). Error bars, standard deviation from >3 independent experiments.', 'hash': 'cdc1930ac4d5ba542cbd0dd5423b9a2c89341b4f94733b58e3e65bc49ef759dd'}, {'image_id': 'nihms100611f4', 'image_file_name': 'nihms100611f4.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f4.jpg', 'caption': 'Effects of disrupting Nudel or dynein on spindle assembly and LB3 localization in the AurA-bead based assay. (a) Progression of spindle morphogenesis and LB3 assembly in mock-depleted egg extracts. LB3 was found along MTs of early asters. As early asters transformed into late aster (white arrows and arrowheads point to bright MT cores and astral MTs, respectively), meshworks of LB3 were seen to surround the late asters. High levels of LB3 were also found throughout the MT balls and spindles. The bright green staining of the beads was caused by the rabbit secondary antibody that recognized anti-AurA antibodies coated on the beads. Yellow arrows point to the LB3 network surrounding the late aster, MT ball, and spindle. (b) Progression of MT and LB3 assembly in Nudel-depleted egg extracts. Formation of early and later asters and the accumulation of γ-tubulin on the AurA-beads occurred normally. However, there was a lack of MT ball and spindle assembly in the absence of Nudel. White arrows and arrowheads point to MT cores and astral MTs, respectively. There was a diminished LB3 network surrounding the late asters as compared to the late aster in the control (compare yellow arrows in the late asters in a and b). (c) Quantification of MT structures at different time points and treatment conditions. All images were acquired on a Leica SP5 confocal microscope with laser lines used indicated in parentheses. Scale, magnetic beads (2.8 µm in diameter). Error bars, standard deviation from >3 independent experiments.', 'hash': 'b0310d267e3d4c1d6b13cdab61251e0d2c7c944978f17b72b460a2a725b8e6b4'}, {'image_id': 'nihms100611f5', 'image_file_name': 'nihms100611f5.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f5.jpg', 'caption': 'Requirement of Nudel in MT organization and LB3 assembly during spindle morphogenesis induced by sperm. (a) Time course of spindle assembly induced by sperm chromatin. Sperm asters assembled in the first 5’–10’ had bright MT cores (white arrow) attached to long astral MT arrays (white arrowhead). The astral arrays underwent polarization toward sperm chromatin to form half spindles in 20–30’, which further organizing into bipolar spindles between 45–120’. (b and c) Localization of Nudel (b) and LB3 (c) on the demembranated sperm. (d–f) Effects of Nudel depletion on MT organization and LB3 accumulation during spindle assembly. Shown are MT, LB3, and sperm chromatin at 10’ (d), 20’ (e), and 60’ (f). Nudel depletion caused disorganization of MT asters (d), polarized MT structures (e), and spindle poles (f) (compare yellow and white arrows). Although LB3 was found throughout the disorganized asters formed in the first 10’ in the absence of Nudel (compare white and yellow arrowheads in d), there was a clear reduction of LB3 on MTs by 20’ (e) and 60’ (f) as compared to those in mock-depleted or Nudel-rescued egg extracts (compare yellow and white arrowheads in e and f). MT structures were quantified in the graphs below the images. DAPI (blue) stained the sperm DNA. All images were acquired on a Leica SP5 confocal microscope. Scale bar, 10 µm. Error bars, standard deviation from >3 independent experiments.', 'hash': '7d21f785dca832f951a863c442f5415480a3db92ed0bf853873e6886e03c495e'}, {'image_id': 'nihms100611f2', 'image_file_name': 'nihms100611f2.jpg', 'image_path': '../data/media_files/PMC2699591/nihms100611f2.jpg', 'caption': 'Identification of Nudel and dynein in the lamin B spindle matrix. (a) Isolation and characterization of the mitotic spindle matrix. AurA-beads were used to stimulate MT assembly in the presence of RanGTP. MT structures (Spindle+aster) were retrieved using a magnet. After depolymerizing MTs using nocodazole and XB buffer washes, the matrix associated with beads in RanGTP reactions was either directly analyzed on SDS-PAGE (Matrix), or treated with 0.5% Triton X100 (Matrix+TX100) and analyzed. Matrix failed to assemble when MT assembly was stimulated by DMSO and then depolymerized (DMSO). Western blotting analyses showed that the isolated matrix retained lamin B3 (LB3), Nudel, and dynein. In contrast, vast majority of TPX2, and tubulin were removed from the matrix after MT depolymerization. (b) Localization of Nudel and LB3 on spindles. Nudel and LB3 were detected using rabbit anti-Nudel and mouse anti-LB3 antibodies, respectively. (c) Localization of dynein and LB3 on spindles. Dynein and LB3 were detected using the mouse monoclonal antibody 74.1 (detecting the dynein intermediate chain) and the rabbit anti-LB3 antibody, respectively. (d and e) Localization of Nudel, LB3, and dynein on the spindle matrix. Images were acquired using a confocal microscope (Leica SP5). Rhodamine tubulin was used to detect MTs in b–e and numbers in parentheses indicate laser lines used. Scale, magnetic beads (2.8 µm in diameter).', 'hash': '862344b6652645a9e98668701e5f38913b20b80b7a1fe0213ba528b30e6af2b2'}] | {'nihms100611f1': ['We found that epitope tagged human lamin B1 and Nudel reciprocally immunoprecipitated each other from HEK293T cells in the presence of detergent NP-40 (Fig. S1). Rabbit polyclonal antibodies that recognized Xenopus Nudel, which shares ~80% amino acid identity with human and mouse Nudel (<xref ref-type="fig" rid="nihms100611f1">Fig. 1a</xref>; ; Fig. S2), could immunoprecipitate LB3 in egg extracts 8, 13 in the presence of detergent (<xref ref-type="fig" rid="nihms100611f1">Fig. 1b</xref>). LB3 antibodies immunoprecipitated dynein in a detergent-insensitive manner as judged by antibodies to dynein intermediate light chains 70.1 (). LB3 antibodies immunoprecipitated dynein in a detergent-insensitive manner as judged by antibodies to dynein intermediate light chains 70.1 (<xref ref-type="fig" rid="nihms100611f1">Fig. 1c</xref>) or 74.1 (data not shown). It was, however, difficult to detect Nudel because it migrated just below the antibody heavy chain. Since similar immunoprecipitation results were obtained in the presence of nocodazole where neither LB3 nor Nudel antibody pulled down tubulin () or 74.1 (data not shown). It was, however, difficult to detect Nudel because it migrated just below the antibody heavy chain. Since similar immunoprecipitation results were obtained in the presence of nocodazole where neither LB3 nor Nudel antibody pulled down tubulin (<xref ref-type="fig" rid="nihms100611f1">Fig. 1d</xref>), LB3, Nudel, and dynein appear to interact with one another independent of tubulin or MTs.), LB3, Nudel, and dynein appear to interact with one another independent of tubulin or MTs.', 'Using purified 6His-tagged full-length Nudel (6His-Nudel, 1–345 aa), N- (6His-N-Nudel, 1–201 aa) and C-terminus (6His-C-Nudel, 167–345 aa) of Nudel (<xref ref-type="fig" rid="nihms100611f1">Fig. 1e</xref>; ; Fig. S3), we found that GST-tagged full length LB3 pulled down 6His-Nudel (<xref ref-type="fig" rid="nihms100611f1">Fig. 1f</xref>). All intermediate filament proteins contain a central α-helical-rod domain flanked by non-α-helical N- and C-terminal regions of variable lengths. The rod domain consists of two coiled-coil regions, coil1 and coil2. The coil2 is conserved among all intermediate filaments ). All intermediate filament proteins contain a central α-helical-rod domain flanked by non-α-helical N- and C-terminal regions of variable lengths. The rod domain consists of two coiled-coil regions, coil1 and coil2. The coil2 is conserved among all intermediate filaments 14. We found that Nudel exhibited stronger interaction with GST-LB3-rod than GST-LB3T (<xref ref-type="fig" rid="nihms100611f1">Fig. 1g</xref>; ; Fig. S3). The coil2 region of LB3-rod appeared to mediate the interaction with Nudel (<xref ref-type="fig" rid="nihms100611f1">Fig. 1h</xref>). The C-terminus of Nudel mediated the interaction with the conserved coil2 of LB3 (). The C-terminus of Nudel mediated the interaction with the conserved coil2 of LB3 (<xref ref-type="fig" rid="nihms100611f1">Fig. 1i; j</xref>). Biosensor binding studies revealed that the equilibrium dissociation constant between Nudel and the coil2 domain of LB3 is ~50 µM (see ). Biosensor binding studies revealed that the equilibrium dissociation constant between Nudel and the coil2 domain of LB3 is ~50 µM (see Supplementary Information).'], 'nihms100611f2': ['To examine whether Nudel and dynein are components of the lamin B spindle matrix, we isolated the matrix using Aurora A (AurA)-bead based assay15 and subjected the matrix to protein sequencing. LB3 and a number of spindle assembly factors (SAF) including dynein were identified in the matrix but not Nudel (Supplementary Information, Fig. S4 and Table S1). Since proteomic analyses are not quantitative and may miss or falsely identify proteins, we also analyzed the isolated matrix using immunoblotting to assess the relative retention of dynein and Nudel on the matrix as compared to tubulin, TPX2, and LB3. Consistent with our earlier findings 8, the isolated matrix was completely disassembled in the presence of detergent (Triton X100). The matrix failed to assemble in the absence of RanGTP even when there was robust MT assembly as stimulated by DMSO. Both dynein and Nudel were present on the isolated matrix and they exhibited similar retention as LB3, whereas the vast majority of tubulin and TPX2 were not retained (<xref ref-type="fig" rid="nihms100611f2">Fig. 2a</xref>). Immunofluorescence analyses revealed that both dynein and Nudel exhibited similar distributions on spindles and spindle matrices to LB3 (). Immunofluorescence analyses revealed that both dynein and Nudel exhibited similar distributions on spindles and spindle matrices to LB3 (<xref ref-type="fig" rid="nihms100611f2">Fig. 2b–e</xref>). Therefore, both dynein and Nudel are components of the lamin B spindle matrix.). Therefore, both dynein and Nudel are components of the lamin B spindle matrix.'], 'nihms100611f3': ['Previous studies of Nudel in mitosis have focused on its kinetochore functions 4, 5, 16. We used the AurA-bead assay, which does not involve chromosomes 15, to test whether Nudel regulates spindle assembly independent of kinetochores. Typically, AurA-beads began to nucleate MT asters between 1’–3’. These early asters had long MT arrays with no obvious bright MT core surrounding the beads (<xref ref-type="fig" rid="nihms100611f3">Fig. 3a</xref>, early aster). The early asters were transformed into late asters between 3’–9’ with increasing MT intensity (, early aster). The early asters were transformed into late asters between 3’–9’ with increasing MT intensity (<xref ref-type="fig" rid="nihms100611f3">Fig. 3a</xref>, compare arrowheads in the early and late asters) and a clear bright MT core appearing around the AurA-beads (, compare arrowheads in the early and late asters) and a clear bright MT core appearing around the AurA-beads (<xref ref-type="fig" rid="nihms100611f3">Fig. 3a</xref>, white dashed circle outlines the bright MT core). Then, the astral array of MTs began to shrink between 6’–11’, which was accompanied by the expansion of the bright MT core surrounding the AurA-beads, resulting in the formation of a ball-like MT structure (, white dashed circle outlines the bright MT core). Then, the astral array of MTs began to shrink between 6’–11’, which was accompanied by the expansion of the bright MT core surrounding the AurA-beads, resulting in the formation of a ball-like MT structure (<xref ref-type="fig" rid="nihms100611f3">Fig. 3a and b</xref>, MT balls are outlined by the white dashed circles in a). Using the ratio between the length of longest astral MT arrays attached to the MT core and the diameter of the bright MT core, we defined that a later aster has a ratio equal or greater than one, whereas a ball-like MT structure has a ratio less than one. Between 13’–15’, most structures are MT balls or spindles and the ratio between the two structures varied, depending on the quality of egg extracts, from 9:1 (poor egg extracts) to 1:1 (good egg extracts) (, MT balls are outlined by the white dashed circles in a). Using the ratio between the length of longest astral MT arrays attached to the MT core and the diameter of the bright MT core, we defined that a later aster has a ratio equal or greater than one, whereas a ball-like MT structure has a ratio less than one. Between 13’–15’, most structures are MT balls or spindles and the ratio between the two structures varied, depending on the quality of egg extracts, from 9:1 (poor egg extracts) to 1:1 (good egg extracts) (<xref ref-type="fig" rid="nihms100611f3">Fig. 3a, b</xref>). For a given egg extract, the number of MT balls and spindles remained similar at 13’ and 15’. Reaction times beyond 15’ often resulted in aggregation of MT structures. Therefore, we focused our analyses up to 15’.). For a given egg extract, the number of MT balls and spindles remained similar at 13’ and 15’. Reaction times beyond 15’ often resulted in aggregation of MT structures. Therefore, we focused our analyses up to 15’.', 'Since overexpressing the N-terminus of NudE in tissue culture cells disrupted mitosis 5, 17, we carried out the AurA-bead assay in the presence of excess GST-N-Nudel and found that both MT ball and spindle assembly were inhibited (<xref ref-type="fig" rid="nihms100611f3">Fig. 3c, d</xref>). In control reactions with GST addition, MT balls or spindles were assembled around AurA-beads by 13’–15’ (). In control reactions with GST addition, MT balls or spindles were assembled around AurA-beads by 13’–15’ (<xref ref-type="fig" rid="nihms100611f3">Fig. 3d</xref>; ; Fig. S5). However, in the presence of GST-N-Nudel, most AurA-beads had late asters with long astral arrays of MTs (yellow arrowheads in <xref ref-type="fig" rid="nihms100611f3">Fig. 3d</xref>) attached to disorganized MT cores (yellow arrow in ) attached to disorganized MT cores (yellow arrow in <xref ref-type="fig" rid="nihms100611f3">Fig. 3d</xref>). Similar to the addition of GST-N-Nudel, depletion of >95% of Nudel resulted in a severe block of assembly of MT balls and spindles, whereas adding back the purified 6His-Nudel fully reversed the defects (). Similar to the addition of GST-N-Nudel, depletion of >95% of Nudel resulted in a severe block of assembly of MT balls and spindles, whereas adding back the purified 6His-Nudel fully reversed the defects (<xref ref-type="fig" rid="nihms100611f3">Fig. 3e</xref>). We found that ). We found that Xenopus Nudel and NudE share 48% overall amino acid identity with each other and they appear to function redundantly in spindle assembly (Supplementary Information; <xref ref-type="fig" rid="nihms100611f3">Fig. 3e</xref> and and Fig. S6a–c) independent of their roles in kinetochores 4,5,16,18.'], 'nihms100611f4': ['We examined whether Nudel and dynein might mediate the MT-dependent assembly of lamin B spindle matrix 8. Indeed, LB3 was localized along the astral MT arrays of the early AurA-asters (<xref ref-type="fig" rid="nihms100611f4">Fig. 4a</xref>). A network of LB3 surrounded the MTs by the late aster stage (). A network of LB3 surrounded the MTs by the late aster stage (<xref ref-type="fig" rid="nihms100611f4">Fig. 4a</xref>, yellow arrows on the late aster). As astral arrays of MTs shrank, LB3 was enriched on MT structures similar to γ-tubulin (, yellow arrows on the late aster). As astral arrays of MTs shrank, LB3 was enriched on MT structures similar to γ-tubulin (<xref ref-type="fig" rid="nihms100611f4">Fig. 4a</xref>). Depletion of Nudel not only blocked assembly of MT balls and spindles as expected but also reduced the intensity of the LB3 network surrounding the late asters (). Depletion of Nudel not only blocked assembly of MT balls and spindles as expected but also reduced the intensity of the LB3 network surrounding the late asters (<xref ref-type="fig" rid="nihms100611f4">Fig. 4b</xref>, compare the yellow arrows on the late asters in a and b). Addition of purified 6His-Nudel rescued the transition of late asters into MT balls and spindles (, compare the yellow arrows on the late asters in a and b). Addition of purified 6His-Nudel rescued the transition of late asters into MT balls and spindles (<xref ref-type="fig" rid="nihms100611f4">Fig. 4c</xref>) as well as the LB3 network surrounding MTs () as well as the LB3 network surrounding MTs (Fig. S7). Dynein inhibition by 70.1 antibody produced similar results to Nudel depletion (<xref ref-type="fig" rid="nihms100611f4">Fig. 4c</xref> and and Supplementary Information).', 'We reasoned that Nudel and dynein could regulate spindle assembly in part by stimulating assembly of lamin B into the spindle matrix. Lamin B in the spindle matrix might in turn help MT organization. If this were the case, one might expect that depleting LB3 would result in MT disorganization in spindles, but the phenotype would be less severe as compared to disrupting either Nudel or dynein. Using AurA-bead assay, we found that unlike disrupting the function of Nudel or dynein, which blocked both MT ball formation and spindle assembly (<xref ref-type="fig" rid="nihms100611f4">Fig. 4</xref>), depleting LB3 still allowed formation of MT balls and bipolar spindles (), depleting LB3 still allowed formation of MT balls and bipolar spindles (<xref ref-type="fig" rid="nihms100611f6">Fig. 6c</xref>). However, these MT structures were more disorganized than controls. Moreover, the MT balls formed in the absence of LB3 had a bigger diameter than those formed in control reactions (). However, these MT structures were more disorganized than controls. Moreover, the MT balls formed in the absence of LB3 had a bigger diameter than those formed in control reactions (<xref ref-type="fig" rid="nihms100611f6">Fig. 6d</xref>). The bipolar spindles assembled in the absence of LB3 were also more elongated than those in controls (). The bipolar spindles assembled in the absence of LB3 were also more elongated than those in controls (<xref ref-type="fig" rid="nihms100611f6">Fig. 6e</xref>).).'], 'nihms100611f5': ['Next, we examined the effect of Nudel on LB3 assembly during spindle morphogenesis induced by demembranated Xenopus sperm. Xenopus sperm stimulated a consistent transformation of astral MT arrays into bipolar spindles. In the first 5’–10’ of reactions, the MT asters induced by sperm had bright MT cores attached to radial array of MTs (<xref ref-type="fig" rid="nihms100611f5">Fig. 5a</xref>), which began to polarize toward the sperm chromatin from 10’–20’, and by ~30’ most MT asters became half spindles (), which began to polarize toward the sperm chromatin from 10’–20’, and by ~30’ most MT asters became half spindles (<xref ref-type="fig" rid="nihms100611f5">Fig. 5a</xref>). By 45’–120’, depending on the quality of the egg extracts, 40–80% of sperm chromatin was associated with bipolar spindles (). By 45’–120’, depending on the quality of the egg extracts, 40–80% of sperm chromatin was associated with bipolar spindles (<xref ref-type="fig" rid="nihms100611f5">Fig. 5a</xref>).).', 'The Xenopus sperm contained both Nudel and LB3 (<xref ref-type="fig" rid="nihms100611f5">Fig. 5b, c</xref>), which may partially compensate for the effect of Nudel depletion from the egg extracts. We found that depleting Nudel did not affect the assembly of MT asters in the first 5’ (data not shown). The asters, however, became disorganized between 5’–15’ (), which may partially compensate for the effect of Nudel depletion from the egg extracts. We found that depleting Nudel did not affect the assembly of MT asters in the first 5’ (data not shown). The asters, however, became disorganized between 5’–15’ (<xref ref-type="fig" rid="nihms100611f5">Fig. 5d</xref>). By 20’, while the control asters accumulated LB3 network throughout the MT arrays, asters assembled in the absence of Nudel had a diminished LB3 network along the MT arrays (compare white and yellow arrowheads in ). By 20’, while the control asters accumulated LB3 network throughout the MT arrays, asters assembled in the absence of Nudel had a diminished LB3 network along the MT arrays (compare white and yellow arrowheads in <xref ref-type="fig" rid="nihms100611f5">Fig. 5e</xref>). Although the MT arrays were able to polarize toward the sperm between 20’–30’, they were not focused at their minus ends (compare white and yellow arrows in ). Although the MT arrays were able to polarize toward the sperm between 20’–30’, they were not focused at their minus ends (compare white and yellow arrows in <xref ref-type="fig" rid="nihms100611f5">Fig. 5e</xref>). By 45’–120’, these structures further developed into fence-like parallel MT arrays around the chromatin (unfocused spindles) with diminished LB3 network on the MTs as compared to control spindles (). By 45’–120’, these structures further developed into fence-like parallel MT arrays around the chromatin (unfocused spindles) with diminished LB3 network on the MTs as compared to control spindles (<xref ref-type="fig" rid="nihms100611f5">Fig. 5f</xref>, compare white and yellow arrows and arrowheads). Both defects of MT organization and diminished accumulation of LB3 could be fully rescued by the purified Nudel (, compare white and yellow arrows and arrowheads). Both defects of MT organization and diminished accumulation of LB3 could be fully rescued by the purified Nudel (<xref ref-type="fig" rid="nihms100611f5">Fig. 5d–f</xref>).).'], 'nihms100611f6': ['To assess the role of Nudel and dynein in lamin B matrix assembly, we prepared the matrix in the presence or absence of Nudel. Whereas depleting Nudel inhibited assembly of matrices containing LB3, addition of purified Nudel largely rescued the defects (graph in <xref ref-type="fig" rid="nihms100611f6">Fig. 6a</xref>). Any matrices formed in Nudel-depleted egg extracts were small as compared to controls, and many AurA-beads were not associated with any matrices (images in ). Any matrices formed in Nudel-depleted egg extracts were small as compared to controls, and many AurA-beads were not associated with any matrices (images in <xref ref-type="fig" rid="nihms100611f6">Fig. 6a</xref>). A similar lack of LB3-matrix assembly was seen when dynein was inhibited by 70.1 antibody (data not shown). We have shown previously that depleting LB3 prevented association of Eg5 with the spindle matrix, but depleting Eg5 or XMAP215 still allowed assembly of matrices containing LB3 ). A similar lack of LB3-matrix assembly was seen when dynein was inhibited by 70.1 antibody (data not shown). We have shown previously that depleting LB3 prevented association of Eg5 with the spindle matrix, but depleting Eg5 or XMAP215 still allowed assembly of matrices containing LB3 8. Consistent with this, we found that inhibiting Eg5 using Monastrol 19 did not affect the assembly of matrices containing LB3, Nudel, or dynein (data not shown). Importantly, depleting LB3 still resulted in assembly of matrices containing Nudel and dynein (<xref ref-type="fig" rid="nihms100611f6">Fig. 6b</xref>). Like dynein ). Like dynein 11, Nudel is localized to the nuclear envelope during prophase in tissue culture cells (Fig. S8). Therefore, Nudel and dynein are in the close proximity of lamin B at the onset of mitosis, which may facilitate their interaction with lamin B after nuclear envelope breakdown to regulate assembly of the lamin B spindle matrix.'], 'nihms100611f7': ['Next, we analyzed the effect of LB3 depletion on MT organization during spindle morphogenesis stimulated by sperm chromatin. Similar to our earlier observation, depleting LB3 caused a reduction of normal spindle assembly. The majority of defective MT structures were multipolar spindles, half spindles, or asters (<xref ref-type="fig" rid="nihms100611f7">Fig. 7</xref>). As compared to Nudel depletion (see ). As compared to Nudel depletion (see <xref ref-type="fig" rid="nihms100611f5">Fig. 5</xref>), removing LB3 from the egg extracts caused less severe defects in minus-end MT focusing. Assembly of MT asters appeared normal in the absence of LB3 before 15’ (), removing LB3 from the egg extracts caused less severe defects in minus-end MT focusing. Assembly of MT asters appeared normal in the absence of LB3 before 15’ (<xref ref-type="fig" rid="nihms100611f7">Fig. 7a</xref>), whereas MT asters became highly disorganized after 5’ when Nudel was depleted (), whereas MT asters became highly disorganized after 5’ when Nudel was depleted (<xref ref-type="fig" rid="nihms100611f5">Fig. 5d</xref>). After 15’ in the absence of LB3, MT structures became disorganized with less MTs polarizing toward the sperm chromatin than that of mock-depletion (compare white and yellow arrows and arrowheads in ). After 15’ in the absence of LB3, MT structures became disorganized with less MTs polarizing toward the sperm chromatin than that of mock-depletion (compare white and yellow arrows and arrowheads in <xref ref-type="fig" rid="nihms100611f7">Fig. 7b</xref>). However, comparing to Nudel depletion at the same time point, the polarized MTs were less splayed at the minus ends (compare ). However, comparing to Nudel depletion at the same time point, the polarized MTs were less splayed at the minus ends (compare <xref ref-type="fig" rid="nihms100611f7">Fig. 7b</xref> to to <xref ref-type="fig" rid="nihms100611f5">Fig. 5e</xref>). Although depletion of LB3 disrupted spindle assembly (). Although depletion of LB3 disrupted spindle assembly (<xref ref-type="fig" rid="nihms100611f7">Fig. 7c</xref>), instead of having completely unfocused spindle poles as seen in Nudel depletion (), instead of having completely unfocused spindle poles as seen in Nudel depletion (<xref ref-type="fig" rid="nihms100611f5">Fig. 5f</xref>), defective spindles formed in the absence of LB3 had partially focused but often multiple poles (), defective spindles formed in the absence of LB3 had partially focused but often multiple poles (<xref ref-type="fig" rid="nihms100611f7">Fig. 7c</xref>).).']} | A Requirement of Nudel and Dynein for Assembly of the Lamin B Spindle Matrix | [
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"dynein",
"mitosis",
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] | Nat Cell Biol | 1236499200 | [{'@Label': 'BACKGROUND', '@NlmCategory': 'BACKGROUND', '#text': 'With the large number of aging individuals requiring screening of cognitive functions for dementing illnesses, there is a necessity for innovative evaluation approaches. One domain that should allow for online, at a distance, examination is speech and language dysfunction, if the auditory and visual transmission is of sufficient quality to allow adequate patient participation and reliable, valid interpretation of signs and symptoms (Duffy et al 1997).'}, {'@Label': 'OBJECTIVE', '@NlmCategory': 'OBJECTIVE', '#text': "Examine the effectiveness of language assessment in mild Alzheimer's patients using telemedicine (TM) compared with traditional in-person (IP) assessment."}, {'@Label': 'DESIGN', '@NlmCategory': 'METHODS', '#text': "Ten patients with mild Alzheimer's disease, enrolled at a Geriatric Memory Clinic received a battery of standard language tests under two conditions: face-to-face and via satellite TM."}, {'@Label': 'RESULTS', '@NlmCategory': 'RESULTS', '#text': 'Comparison of TM and IP testing conditions were assessed within each for scores on each test in the two conditions. On each of the five language tasks, the Wilcoxon signed ranks test indicated no significant difference on performance between the TM and IP conditions for each participant. Overall acceptance of the TM evaluation in an elderly population was rated at a high level except for one individual.'}, {'@Label': 'CONCLUSION', '@NlmCategory': 'CONCLUSIONS', '#text': 'Telemedicine can improve access to speech and language evaluation services which is relevant to both dementia and other neurological diseases of the elderly. In particular, this specific assessment tool can be used to provide evaluations in under-served rural areas.'}] | [
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"Language Tests",
"Male",
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