BIX-01294 reduces USP9X level in the concentration- and time-dependent manner, and USP9X knockdown enhances MCL1 degradation and apoptosis. 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). After control siRNA and USP9X siRNA transfection, cells were treated with 10 μmol/l of BIX-01294 for 24 hours. We used antibodies against USP9X, MCL1, CASP9, CASP3, PARP1 and ACTB for western blot analysis (C) or used Annexin V-FITC Apoptosis Detection Kit for detection of apoptotic cells in T24 (D) and 5637 (E). In D and E, 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).