Figure 3. Caspase-9 inhibitor blocked the activation of caspase-3 induced by BBMD3 and effects of BBMD3 on regulatory proteins of apoptosis in osteosarcoma cells. (A) KHOS cells were first treated with 20 µM csapase-8 inhibitor (Z-IEHD-FMK) or caspase-9 inhibitor (Z-LEHD-FMK) for 30 min, and then 5 µM BBMD3 was added to cells for another 24 h. Expression of cleaved caspase-3 and PARP was analyzed by immunoblotting assays. (B) BBMD3 increased cleaved caspase-9 and in KHOS and G292 cells. (C) BBMD3 increased release of cytochrome c to cytosol in KHOS and G292 cells. Cells were treated with 5 µM BBMD3 for 24 h and cytosol was isolated. Cytochrome c was detected by immunoblotting assays. Effects of BBMD3 on anti-apoptotic proteins (D) and pro-apoptotic proteins (E) in KHOS, G292, and MG-63 cells were determined by immunoblotting assays after 24 h treatment.