FIG. 6.

Depletion of Bax by RNA interference blocks p53-induced Cyt c release from isolated mitochondria, and p53 can promote Bax insertion into mitochondrial membranes. (A) The pSilencer 2.1-U6 hygro plasmids bearing the Bax hairpin insert or a control hairpin insert were transfected into H1299 p53-null cells using Lipofectamine 2000. The levels of Bax expression were analyzed by Western blotting using a Bax antibody. (B) H1299 cells expressing Bax siRNA or control siRNA were treated with cisplatin (20 μM) for 48 h. Cell viability was assessed as described in the legend for Fig. 1C. (C) Intact mitochondria were isolated from H1299 cells expressing Bax siRNA or vector control and incubated with increasing concentrations of purified p53 protein in MSB buffer at 30°C for 30 min. Alkali extraction of Bax using 0.1 M Na₂CO₃ (pH 11.5) was performed as described in Materials and Methods. The alkali-resistant Bax (i.e., nonextractable) was determined by Western blotting using a Bax antibody. (D) Intact mitochondria were isolated from p53-null human H1299 cells expressing Bax siRNA or vector control and incubated with increasing concentrations of purified p53 protein in MSB buffer at 30°C for 30 min. Cyt c release was analyzed as described in the legend for Fig. 5D.