BIX-01294 up-regulates the level of DDIT3, and DDIT3 knockdown suppresses the apoptosis induced by BIX-01294 in human 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). T24 and 5637 cells were pretreated with 4-PBA-Na+ (1 mM) for 30 min and then cotreated with BIX-01294 (10 μM) for another 24 h. Levels of protein expression were analyzed by western blot using antibodies against DDIT3, CASP9, CASP3 and ACTB (C). After control siRNA and DDIT3 siRNA transfection, cells were treated with 10 μmol/l of BIX-01294 for 24 hours. Whole-cell protein lysates were harvested for Western blot analysis by using antibodies against DDIT3, PARP1, PMAIP1, MCL1 and ACTB (D). T24 (E) and 5637 (F) cells were transfected with control siRNA and DDIT3 siRNA for 48 hours. After treatment with BIX-01294 (10 μmol/l) for 24 h, cells were stained with Annexin V-FITC/PI and detected by flow cytometry analysis. In E and F, the early apoptosis detected by flow cytometry is the upper one and the total apoptosis is the lower one. 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).