FIGURE 3.

Effect of KS18 on apoptosis regulators. (A), The schematic presentation of apoptotic regulators. In the indirect activation, upregulation of BH3-only proteins will act as inhibitors of anti-apoptotic proteins by competing for their binding with Bax and Bak proteins, leading Bax and Bak to oligomerize. In the direct activation, upregulation of BH3 activators proteins directly activates Bax and Bak. (B), U266 MM cells were treated with or without KS18 (5 µM) for 24 h. (C, D), an immunoprecipitation test was conducted to evaluate the interaction between Mcl-1 and Bim, Bak, and Bax, utilizing either anti-Mcl-1 or anti-Bim for the pull-down. Immunoprecipitation followed by western blotting was conducted as outlined in the Materials and Methods section. (E–G), U266 MM cells were treated with the indicated doses of KS18 for 24 h. Specific to section (F), after incubation the cytosolic fractions were isolated using a cytochrome C kit. (H, I), Caspases 3/7 and Annexin V staining were used to identify apoptotic cells. U266 cells were treated with KS18 (5 µM) for 24 h before being stained with caspases 3/7 dye or Annexin V dye as described in Materials and Methods section and evaluated using the Muse^® Cell Analyzer. The total number of apoptotic cells was enumerated for samples (n = 3) and unpaired t test was performed using GraphPad prism software. In all experiments, vehicle treated cells served as control. For sections (B–G), after incubation, the cells were lyzed and immunoblotting was performed using the mentioned antibodies. **P ≤ 0.01, ****P ≤ 0.0001.