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Apoptosis induced by selective BH3-mimetics
ABC
DE
Figure 6. BH3-mimetic-induced cell death is mediated by BAX. (A-D) Knockdown of BAX using short-inter- fering (si)RNA-mediated gene silenc- ing was performed before treatment with ABT-199 (A) in RIVA (3 nM) or U2932 (10 nM) cells, upon treatment with A1331852 (B) in RCK8 (3 nM) or SUDHL8 (10 nM) cells, and upon treatment with S63845 (C) in SUDHL10 (100 nM) or U2946 (300 nM) cells for 4 h and phosphatidylser- ine (PS) exposure was analyzed by staining with annexin V-FITC and flow cytometry. Untransfected cells and non-targeting siRNA (siCtrl) were used as controls. The mean and standard deviation (SD) are shown (n=3-4). (D) Knockdown efficiency was assessed by western blotting. (E) Knockdown of BAX with siRNA constructs #1 and #3 combined was followed by treatment with ABT-199 in RIVA (10 nM) or U2932 (30 nM) cells for 4 h and analysis of BAK activation by flow cytometry. The mean and SD are shown (n=3 for RIVA and n=2 for U2932). (F) The interaction between BAK and BAX was assessed by immunoprecipitation (IP) in RIVA or U2932 cells with or without treatment with ABT-199 (+;-). Input lanes show the presence of overall protein in the lysate, and IP lanes show the interac- tion between BAX and BAK upon treat- ment with ABT-199. Protein G beads without primary antibody were used to control for unspecific binding. *indicates IgG band. GAPDH was used as a loading control. *P<0.05;**P<0.01; ***P<0.001.
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