human bladder epithelial cell line 5637  (ATCC)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RBM15 drives bladder cancer progression through YTHDF2-dependent m6A-mediated regulation of ZO2

doi: 10.1186/s13046-026-03684-9

Figure Lengend Snippet: RBM15 drives m6A hypermethylation and the malignant progression of BC in vitro. A , B qPCR ( A ) and WB ( B ) analyses showing upregulated RBM15 expression in a panel of BC cell lines compared with the immortalized urothelial cell line SV-HUC-1. C , D Efficient knockdown of RBM15 in T24 and 5637 cells using two independent shRNAs (shRBM15-1 and shRBM15-2), as confirmed by WB ( C ) and qPCR ( D ). E RNA dot blot analysis showing a reduction in global m6A methylation levels upon RBM15 knockdown. Methylene blue (MB) staining served as a loading control. F , G CCK-8 assays showing that RBM15 knockdown significantly inhibited the proliferation of T24 ( F ) and 5637 ( G ) cells. H , I Transwell assays demonstrating that RBM15 knockdown suppressed the migration and invasion of T24 ( H ) and 5637 ( I ) cells. Representative images and the results of the quantitative analysis are shown. Scale bar, 100 × (10× objective × 10× ocular). Three randomly selected fields per sample (100× magnification) were quantified. J , K WB ( J ) and qPCR ( K ) confirmation of successful RBM15 re-expression (RBM15res) in RBM15-knockdown cells. L RNA dot blot analysis showing that the re-expression of RBM15 rescued global m6A methylation levels. M , N CCK-8 assays showing that RBM15 re-expression reversed the inhibition of the proliferation of T24 ( M ) and 5637 ( N ) cells. O , P Transwell assays showing that RBM15 re-expression restored the migratory and invasive capacities of T24 ( O ) and 5637 ( P ) cells. Scale bars are the same as in H–I. ** p < 0.01 and *** p < 0.001

Article Snippet: Human 293T, T24, and 5637 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Expressing, Knockdown, Dot Blot, Methylation, Staining, Control, CCK-8 Assay, Migration, Inhibition

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RBM15 drives bladder cancer progression through YTHDF2-dependent m6A-mediated regulation of ZO2

doi: 10.1186/s13046-026-03684-9

Figure Lengend Snippet: RBM15 drives the EMT by regulating the expression and nuclear localization of ZO2. A , B qPCR ( A ) and WB ( B ) analyses of RBM15 and ZO2 expression in T24 cells following RBM15 knockdown by shRNA. C , D Rescue of ZO2 expression was assessed by qPCR ( C ) and WB ( D ) in RBM15-knockdown T24 cells re-expressing RBM15 (shRBM15 + Vector vs. shRBM15 + RBM15res). E Gene set enrichment analysis (GSEA) plots showing the significant negative enrichment of pathway related to the extracellular matrix receptor interaction in RBM15-knockdown cells. F WB analysis of EMT-related markers in T24 and 5637 cells after RBM15 knockdown. G WB analysis of EMT markers in RBM15-knockdown T24 and 5637 cells following the re-expression of RBM15, indicating that Vimentin and Snail are the primary EMT-related proteins regulated by RBM15. H IF staining for ZO2 (green) in T24 cells after RBM15 knockdown. Nuclei were counterstained with DAPI (blue). I Nuclear and cytoplasmic fractions from RBM15-knockdown cells were subjected to WB to detect ZO2 protein levels. LAMB1 and GAPDH served as nuclear and cytoplasmic loading controls, respectively. J ChIP-qPCR validation of ZO2 enrichment at the SNAI1 promoter. IgG was used as a negative control, and a known nonbinding region served as a negative control region. K WB analysis of RBM15, ZO2, and Snail protein expression in T24 cells with single or double knockdown of RBM15 and/or ZO2. L WB analysis of RBM15, ZO2, and Snail protein expression in T24 cells with single or dual overexpression of RBM15 and/or ZO2. M Schematic model illustrating the proposed mechanism. The RBM15-mediated m6A modification promotes ZO2 mRNA degradation. Despite the global reduction in ZO2 levels, nuclear accumulation of ZO2 is paradoxically increased, and ZO2 then binds to the Snail promoter, leading to increased Snail expression and facilitating the epithelial-mesenchymal transition. *** p < 0.001

Article Snippet: Human 293T, T24, and 5637 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Knockdown, shRNA, Plasmid Preparation, Staining, ChIP-qPCR, Biomarker Discovery, Negative Control, Over Expression, Modification