Fig. 2

MCL-1 down-regulation and activation of BAX and BAK are required for S63845/SKI-606-mediated apoptosis. a U937 cells were exposed to the indicated concentrations of S63845 or MIK665 ± SKI-606 for 24 h, after which the level of MCL-1 was determined by western blot analysis. Numerals under the blots correspond to densitometric readings normalized to untreated controls (1.0). b, c Ectopic expression of MCL-1 in U937 cells. b Cells were treated (24 h) with 20 nM S63845 ± 2 µM SKI-606 and cell death was determined by 7-AAD staining and flow cytometric analysis (n = 4 in each group). Inset, levels of MCL-1 by western blot after overexpression. c Western blot analysis of PARP, cleaved- PARP, as well as cleaved-Caspase-3 was performed. β-actin was assayed to ensure equivalent loading and transfer. d U937 cells were exposed to S63845 (20 nM) and SKI-606 (2 μM) alone or in combination for 24 h, after which subcellular fractions were obtained and subjected to western blot analysis to monitor the release of cytochrome c, BAK, and BAX into the cytosol. S-100, cytosol; Cyto c = cytochrome c. Numerals under the blots correspond to densitometric readings normalized to untreated controls (1.0). e U937 cells were exposed to S63845 ± SKI-606 for 24 h, after which cells were lysed in buffer containing 1% CHAPS; conformationally changed BAX and BAK proteins were immunoprecipitated using anti-BAX 6A7 and anti-BAK Ab1 antibodies, respectively, and subjected to western blot analysis using polyclonal BAX or BAK antibodies. f Following 24 h treatment, U937 cells were lysed in buffer and immunoprecipitated (IP) using anti-BAK antibody, followed by western blot analysis using anti-BAK, anti-BAX, or anti-MCL-1 antibodies as indicated. For all IP assay, IPs without cell lysate (-lysate) and/or with IgG (instead of primary antibodies) were carried out as controls; Input lysates were also subjected to western blot analysis to monitor relative protein levels. IgG levels are shown to ensure equal loading of IP antibodies. g BAK and/or BAX CRISPR knockout U937 cells were incubated with S63845 ± SKI-606 for 24 h. Cell death was determined using 7-AAD staining and flow cytometry. Values represent the mean % ± SD for three separate experiments. h BAK, BAX, PARP, cleaved-PARP and cleaved-Caspase-3 were detected by western blot. β-actin was assayed to ensure equivalent loading and transfer. CF, cleavage fragment; EV, empty vector; OE, overexpression. ****P < 0.0001; ^####P < 0.0001