Serum-Nutrient Starvation Induces Cell Death Mediated by Bax and Puma That Is Counteracted by p21 and Unmasked by Bcl-xL Inhibition
Figure 2
Puma and Bax as key actors of apoptosis induced by starvation in p21-negative HCT116 cells.
(A) The indicated cells were placed in complete medium (CM) or in starved medium for 24 h (EBSS). Apoptosis was assayed by APO 2.7 staining using flow cytometry. Data are expressed as a percentage of the total population. (B) Mitochondria (M) and S100 (S100) lysates from HCT116 p21−/− cells, starved 16 h in EBSS medium (EBSS) or not (CM), were analysed by western blot analysis for F1ATPase and Cytochrome C (Cyt C). (C) Viability of the indicated cells placed under starvation during 24 h was analyzed by PI staining followed by flow cytometry. (D) The indicated cells were placed under complete medium (CM) or in starved medium (EBSS). After 24 h, protein extracts were prepared and the expression of the indicated protein was analysed by western blotting. The amount of Puma was evaluated in each condition by densitometric analysis from three independent experiments and normalized to the amount of Puma level measured in complete medium in wild-type cells (E) The p21 negative HCT116 cells were transfected with the indicated siRNA. 48 h later, cells were starved in EBSS medium for 24 h. Western blot analysis of Bax, Puma and Bim was performed and cell death was assayed by a trypan blue-staining. Data presented in (A), (C) and (E) are mean ± SEM of three independent experiments.