Figure 1.

MCL-1 is degraded following nutrient deprivation. (A) HeLa cells were incubated for 4 h in either EBSS or media without glucose/pyruvate. Lysates were then analysed by western blot for the presence of MCL-1, as well as actin as a loading control. (B) CGNs were subjected to 17 h hypoxia after 7 days in vitro. Protein lysates were collected at indicated times after reoxygenation and blotted for MCL-1 expression and for actin as a loading control. (C) H1299 cells stably expressing GFP–LC3 were incubated for 2 h in EBSS, and expression of various BCL-2 homologues was analysed by western blot. Alternatively, primary cortical neurons were incubated for 4 h in media without glucose/pyruvate. (D) H1299 cells stably expressing GFP–LC3 were incubated for 3 h in media without glucose/pyruvate, followed by re-addition of glucose and pyruvate for the indicated times. (E) HeLa cells were infected with 5 p.f.u. per cell of either Ad HA-tBID or Ad LacZ for 7 h, the media being changed to EBSS for the last 3 h where indicated. Alternatively, cells were treated with 50 μM camptothecin for 3 h. (F, G) HeLa cells were treated as in E in the presence of 50 μM zVAD-FMK and analysed by immunofluorescence for active BAX (6A7 epitope; F) or cyt c (G). Data are expressed as the average of three experiments±s.d. ^*P<0.01.