Figure 4: Bax is an essential for SMBA induction of apoptosis and Cyt c release.

(a) Levels of Bax were analysed by western blot in wild-type (WT), Bax^−/−, Bak^−/− or Bax/Bak double-knockout (DKO) MEF cells. (b) WT, Bax^−/−, Bak^−/− or DKO MEF cells were treated with high concentration (25 μM) of SMBA1, SMBA2 or SMBA3 for 24 h. Cell viability was analysed by PI-Annexin-V staining with FACS analysis. The error bars indicate±s.d. of three separate experiments. (c) Mitochondria were isolated from WT, Bax^−/−, Bak^−/− or DKO MEF cells. The isolated mitochondria were then treated with lead compound SMBA1 (5 μM) in mitochondrial buffer for 30 min at 30 °C. After centrifugation, Cyt c in supernatant fraction (that is, Cyt c release) and actin in mitochondrial fraction were analysed by western blot (full blots can be found in Supplementary Fig. 8). Ctrl, control.