Figure 8.

Beclin-1 contributes to GX15-070-induced apoptosis but not to autophagy. (a) CEM or Molt-4 cells were infected with lentiviruses expressing shRNAs for either non-targeting control or Beclin-1 (Bec). Puromycin-resistant cells were pooled after each infection. Downregulation of Beclin-1 expression was monitored by western blotting (in sets). Cells were treated with 100 nM GX15-070 for 48 h and the percentage of cell death was determined by Annexin V-PI staining followed by FACS analysis. Values represent the mean±S.D. of three independent experiments. (b) Cells were treated with 100 nM GX15-070 for the indicated times, and LC3 conversion, caspase-3 cleavage, and AIF release were assessed by western blot analyses. (c) CEM or Molt-4 cells were treated with 100 nM GX15-070 for 24 h. Total cell extracts were subjected to immunoprecipitation with anti-Beclin-1. Western blotting was performed on precipitated samples and on lysates collected before immunoprecipitation with Beclin-1, BCL-2, or BCL-X_L antibodies. (d) Reciprocal immunoprecipitation was performed with anti-BCL-2. Western blotting was conducted on precipitated samples and on lysates collected before immunoprecipitation with BCL-2 or Beclin-1 antibodies