umuc3 cell lines  (ATCC)

Journal: Cell Death & Disease

Article Title: PRDM1 restricts bladder cancer progression and enhances chemosensitivity by suppressing OTUD6A-mediated deubiquitination of CDC6

doi: 10.1038/s41419-026-08498-3

Figure Lengend Snippet: A The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes (DEGs). B Gene Ontology (GO) analysis of the DEGs. Dot plots of Biological Process were shown. C GSEA analysis was performed using T24 cells with PRDM1-knockdown and control cells and showed significant enrichment of the “Cell cycle” pathway. D Indicated T24 cells were synchronized in prometaphase and released into fresh medium. Cells were analysed at the indicated time points by flow cytometry. E , F The proliferation of PRDM1-knockdown and control T24 cells was examined by CCK-8 assays ( E ) and EdU incorporation assays ( F ). Scale bars, 20 μm. G, H The proliferation of PRDM1-overexpressing and control T24 cells was examined by CCK-8 assays ( G ) and EdU incorporation assays ( H ). Scale bars, 20 μm. I CCK-8 assays were used to examine the proliferation of the indicated UMUC3 cells. J Growth curves of the PRDM1-knockdown and control T24 tumors are shown. Tumors were measured every 4 days. K An image of subcutaneous tumors formed by the PRDM1-knockdown and control T24 and UMUC3 cells is shown. L The PRDM1-knockdown and control T24 and UMUC3 tumors were weighed. M Representative IHC images indicating Ki-67 and PRDM1 expression in the PRDM1-knockdown and control T24 and UMUC3 tumors are shown. Scale bars, 50 μm (left) and 20 μm (right). N Growth curves of the PRDM1-overexpressing and control T24 tumors are shown. Tumors were measured every 4 days. O An image of subcutaneous tumors formed by the PRDM1-overexpressing and control T24 and UMUC3 cells is shown. P The PRDM1-overexpressing and control T24 and UMUC3 tumors were weighed. Q Representative IHC images indicating Ki-67 and PRDM1 expression in the PRDM1-overexpressing and control T24 and UMUC3 tumors are shown. Scale bars, 50 μm (left) and 20 μm (right). All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. ** P < 0.01, *** P < 0.001, based on two-tailed Student’s t test.

Article Snippet: HEK293T and UMUC3 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11995065).

Techniques: Knockdown, Control, Flow Cytometry, CCK-8 Assay, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRDM1 restricts bladder cancer progression and enhances chemosensitivity by suppressing OTUD6A-mediated deubiquitination of CDC6

doi: 10.1038/s41419-026-08498-3

Figure Lengend Snippet: A , B The cell viability of the PRDM1-overexpressing ( A ) and PRDM1-knockdown ( B ) T24 cells was determined after 48 h of continuous exposure to multiple concentrations of gemcitabine. C The indicated T24 cells were treated with different concentrations of gemcitabine. Cell survival was determined by colony formation assays. Representative images of colony formation assays of the indicated T24 cells treated with different concentrations of gemcitabine are shown (left). D Apoptosis was measured by TUNEL assays in the indicated T24 cells treated with or without gemcitabine for 48 h. E Cleaved caspase-3 protein levels in the indicated T24 and UMUC3 cells treated with or without gemcitabine for 48 h were determined by Western blot. F The γH2A.X protein level was measured by immunofluorescence staining in the indicated T24 cells treated with or without gemcitabine for 48 h. Representative immunofluorescence images are shown (left). Scale bars, 20 μm. G , H The amount of DNA strand breaks was quantified by alkaline comet assays in the indicated T24 and UMUC3 cells treated with or without gemcitabine for 48 h. Representative images are shown (left). Scale bars, 20 μm. I ATR-Chk1 pathway protein levels in the indicated T24 cells treated with or without gemcitabine for 6 h were determined by Western blot. J The effects of ATR inhibitor (VE-821) on regulating the sensitivity of PRDM1-knockdown T24 cells to gemcitabine was determined by CCK-8 assays. K Growth curves of the indicated subcutaneous T24 tumors treated with or without gemcitabine are shown. L The indicated subcutaneous T24 and UMUC3 tumors were weighed. M PRDM1 expression levels were measured in the gemcitabine resistant (GR) and wild-type UMUC3 cells by Western blot. N Growth curves of the indicated subcutaneous UMUC3 GR tumors treated with or without gemcitabine are shown. O The indicated subcutaneous UMUC3 GR tumors were weighed. All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant, based on two-tailed Student’s t test.

Article Snippet: HEK293T and UMUC3 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11995065).

Techniques: Knockdown, TUNEL Assay, Western Blot, Immunofluorescence, Staining, CCK-8 Assay, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRDM1 restricts bladder cancer progression and enhances chemosensitivity by suppressing OTUD6A-mediated deubiquitination of CDC6

doi: 10.1038/s41419-026-08498-3

Figure Lengend Snippet: A The protein levels of PRDM1 and CDC6 in the indicated T24 and UMUC3 cells were determined by Western blot. B , C CCK-8 assays ( B ) and EdU incorporation assays ( C ) were used to examine the proliferation of the indicated T24 cells. Scale bars, 20 μm. D Growth curves of the indicated T24 tumors are shown. Tumors were measured every 4 days. E An image of subcutaneous tumors formed by the indicated T24 and UMUC3 cells is shown. F The indicated T24 and UMUC3 tumors were weighed. G The cell viability of the indicated T24 cells was determined after 48 h of continuous exposure to multiple concentrations of gemcitabine. H Apoptosis was measured by TUNEL assays in the indicated T24 cells treated with or without gemcitabine for 48 h. I The amount of DNA strand breaks was quantified by alkaline comet assays in the indicated T24 cells treated with or without gemcitabine for 48 h. J ATR-Chk1 pathway protein levels in the indicated T24 cells treated with or without gemcitabine for 6 h were determined by Western blot. K An image of subcutaneous tumors formed by the indicated T24 treated with gemcitabine is shown. L The indicated T24 tumors were weighed. M Representative IHC images indicating cleaved caspase-3 and γH2A.X expression in the indicated T24 tumors are shown. Scale bars, 50 μm (left) and 20 μm (right). N PRDM1 and CDC6 expression levels were measured in the GR and wild-type UMUC3 cells by Western blot. O The cell viability of the indicated UMUC3 GR cells was determined after 48 h of continuous exposure to multiple concentrations of gemcitabine. P Apoptosis was measured by TUNEL assays in the indicated UMUC3 GR cells treated with gemcitabine for 48 h. Q Growth curves of the subcutaneous indicated UMUC3 GR tumors treated with gemcitabine are shown. R The indicated UMUC3 GR tumors were weighed. All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant, based on two-tailed Student’s t test.

Article Snippet: HEK293T and UMUC3 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11995065).

Techniques: Western Blot, CCK-8 Assay, TUNEL Assay, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRDM1 restricts bladder cancer progression and enhances chemosensitivity by suppressing OTUD6A-mediated deubiquitination of CDC6

doi: 10.1038/s41419-026-08498-3

Figure Lengend Snippet: A , B PRDM1 and CDC6 expression levels were measured in the PRDM1-overexpressing ( A ) and -knockdown ( B ) T24 and UMUC3 cells by qPCR; the levels in WT cells were set as 1. C , D PRDM1-overexpressing ( C ) and -knockdown ( D ) T24 cells were treated with CHX and harvested at the indicated time points prior to Western blot (left). The intensities of the CDC6 bands were quantified from Western blot analysis (right); the intensity at 0 min was set as 1. E HEK293T cells transfected with the indicated vectors were treated with CHX and harvested at the indicated time points prior to Western blot (left). The intensities of the CDC6 bands were quantified from Western blot analysis (right); the intensity at 0 min was set as 1. F Western blot analysis of cell lysates from the indicated T24 cells treated with MG132 or DMSO for 6 h. G Western blot analysis of cell lysates from the indicated T24 cells treated with chloroquine (CQ) or DMSO for 6 h. H T24 cells transfected with the indicated vectors were treated with MG132 for 6 h. Whole-cell lysates were prepared and subjected to IP with an anti-Myc antibody. The immunoprecipitates were analysed by Western blot. I Flag-PRDM1/Flag vector, Myc-CDC6 was cotransfected with wild-type HA-Ub or its lysine residue mutants (K48 indicates that all lysines except for K48 were mutated to arginine) into T24 cells for 24 h. The cells were treated with MG132 for 6 h. Whole-cell lysates were prepared and subjected to IP with an anti-Myc antibody. The immunoprecipitates were analysed by Western blot. J GSEA analysis was performed using T24 cells with PRDM1-knockdown and control cells and showed significant enrichment of the “protein deubiquitination involved in ubiquitin-dependent protein catabolic process” pathway. All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. *** P < 0.001, n.s. not significant, based on two-tailed Student’s t test.

Article Snippet: HEK293T and UMUC3 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11995065).

Techniques: Expressing, Knockdown, Western Blot, Transfection, Plasmid Preparation, Residue, Control, Ubiquitin Proteomics, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRDM1 restricts bladder cancer progression and enhances chemosensitivity by suppressing OTUD6A-mediated deubiquitination of CDC6

doi: 10.1038/s41419-026-08498-3

Figure Lengend Snippet: A Expression levels of ubiqutination-related protein (OTUD6A, FZR1, CDT2 and Cyclin F) in the PRDM1-knockdown T24 and UMUC3 cells were determined by Western blot. B Indicated T24 cells transfected with Flag-OTUD6A plasmids/empty vectors, Myc-CDC6 and HA-Ub were treated with MG132 for 6 h. Whole-cell lysates were prepared and subjected to IP with an anti-Myc antibody. The immunoprecipitates were analysed by Western blot. C The protein levels of PRDM1, CDC6 and OTUD6A in the indicated T24 and UMUC3 cells were determined by Western blot. D , E CCK-8 assays ( D ) and EdU incorporation assays ( E ) were used to examine the proliferation of the indicated T24 cells. F The indicated T24 tumors were weighed. G Representative IHC images indicating Ki-67, PRDM1, CDC6 and OTUD6A expression in the indicated T24 tumors are shown. H Apoptosis was measured by TUNEL assays in the indicated T24 cells treated with or without gemcitabine for 48 h. I The amount of DNA strand breaks was quantified by alkaline comet assays in the indicated T24 cells treated with or without gemcitabine for 48 h. J ATR-Chk1 pathway protein levels in the indicated T24 cells treated with or without gemcitabine for 6 h were determined by Western blot. K An image of subcutaneous tumors formed by the indicated T24 treated with gemcitabine is shown. L The indicated T24 tumors were weighed. M The protein levels of PRDM1, OTUD6A and CDC6 in the indicated UMUC3 GR cells were determined by Western blot. N Growth curves of the subcutaneous indicated UMUC3 GR tumors treated with gemcitabine are shown. O The indicated UMUC3 GR tumors were weighed. All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. ** P < 0.01, *** P < 0.001, n.s. not significant, based on two-tailed Student’s t test.

Article Snippet: HEK293T and UMUC3 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11995065).

Techniques: Expressing, Knockdown, Western Blot, Transfection, CCK-8 Assay, TUNEL Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PRDM1 restricts bladder cancer progression and enhances chemosensitivity by suppressing OTUD6A-mediated deubiquitination of CDC6

doi: 10.1038/s41419-026-08498-3

Figure Lengend Snippet: A , B CDC6 and OTUD6A expression levels were measured in the PRDM1-overexpressing ( A ) and -knockdown ( B ) T24 and UMUC3 cells by qPCR; the levels in WT cells were set as 1. C The mRNA levels of PRDM1, CDC6 and OTUD6A were determined by RT-qPCR, and the levels in Flag vector transfected HEK293T cells were set as 1. D The enrichment of PRDM1 protein in the OTUD6A promoter region was determined by ChIP assays in T24 cells. E ChIP-seq binding peak for PRDM1 in T24 cells is shown (obtained using IGV software). F ChIP-qPCR was performed to evaluate the enrichment of PRDM1 in the OTUD6A promoter region in PRDM1-overexpressing T24 cells. ChIP enrichment signals were normalized to the input signal. G The relative luciferase activities of OTUD6A promoter fragments containing reporters in PRDM1-overexpressing T24 and UMUC3 cells were examined by dual-luciferase reporter assays. H Schematic of mutated PRDM1 binding site in the OTUD6A promoter region was shown. I The relative luciferase activities of OTUD6A promoter fragments containing the WT and mutated (MUT) PRDM1 binding sites of as reporters in T24 cells transfected with Flag-PRDM1 or empty Flag vector were examined by dual-luciferase reporter assays. All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. ** P < 0.01, *** P < 0.001, n.s. not significant, based on two-tailed Student’s t test.

Article Snippet: HEK293T and UMUC3 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11995065).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Plasmid Preparation, Transfection, ChIP-sequencing, Binding Assay, Software, ChIP-qPCR, Luciferase, Two Tailed Test