A, Schematic of the PCR primers used to detect total GSDMB (F1/R1) or specific isoforms (F2/R2). B, Expression of GSDMB in indicated cell lines, assessed by qRT-PCR using F1/R1 primers, relative to GAPDH. C, PCR products of GSDMB isoforms, resolved on 3% agarose gels, were amplified using F2/R2 primers. D, Effect of IFNγ and methotrexate (Mtx) on total GSDMB expression in SW837 and SW1116 cells, assessed by qRT-PCR with F1/R1 primers. E, Effect of IFNγ and Mtx on the expression of GSDMB isoforms in SW1116 cells, assessed by qRT-PCR with isoform-specific primers. F, Expression of GSDMB isoforms in primary tumors compared to adjacent normal tissue in the Cancer DEIso and TCGA database. Comparisons were calculated by paired Wilcoxon test and P<0.01 was considered significant (highlighted in black boxes). G, Correlation of GSDMB3 expression and survival in BLCA patients. Data in B and D are mean ± s.d. of biological triplicates and are representative of three independent experiments. Statistical analysis was performed using the two-tailed Student’s t-test. **P < 0.01. *P < 0.05.