BIX-01294 up-regulates the expression of PMAIP1 and down-regulates MCL1 in bladder cancer cells. T24 and 5637 cells were treated with indicated concentrations of BIX-01294 for 24 hours or the cells were treated with 10 μmol/l of BIX-01294 for various lengths of time. Both attached and suspended cells were harvested for Western blot analysis (A and B). We used control siRNA and PMAIP1 siRNA to transfect T24 (C and D) and 5637 (C and E) for 48 hours and then added BIX-01294 (10 μmol/l) into culture medium and harvested the cells for western blot analysis by using antibodies against PMAIP1, MCL1, CASP9, CASP3, PARP1, DDIT3 and ACTB or for detection of apoptotic cells using Annexin V-FITC staining. In D and E, the early apoptosis detected by flow cytometry is the upper one and the total apoptosis is the lower one. MCL1, CASP9, CASP3 and ACTB were detected by western blot after T24 and 5637 cells transfected with pcDNA3.1 and pcDNA3.1-MCL1 (F). Columns: mean of triplicate treatments; bars: ± SD. The statistical differences between the two treatments were analyzed by two-sided unpaired Student’s t-tests (*p < 0.05; **p < 0.01; *** p < 0.001).