Effects of 17-AAG on apoptosis signaling in HL-60 cell types. (A) Ectopic overexpression of Bcl-2 or Bcl-xL inhibits 17-AAG—induced Bax conformational change. HL-60/Neo, HL-60/Bcl-2, and HL-60/Bcl-xL cells were treated with 2.0 μM 17-AAG for the indicated time intervals. Following this, using the protein A plus protein G beads coated with anti-6A7 antibody, Bax was immunoprecipitated from the cell lysates. Immunoprecipitates were immunoblotted with anti-Bax antibody. (B) Ectopic overexpression of Bcl-2 or Bcl-xL inhibits 17-AAG—induced cytosolic accumulation of cyt-c and Smac and PARP cleavage activity of caspase-3. HL-60/Neo, HL-60/Bcl-2, and HL-60/Bcl-xL cells were treated with 2.0 μM 17-AAG for 48 hours. Following this, cell lysates were harvested, and Western blot analyses were performed for the full-length PARP (116 kDa) and its cleaved fragments (85 kDa). Alternatively, S100 (cytosolic) or the heavy-membrane fractions (HMFs) were obtained from the harvested cells and used for the immunoblot analyses for cytosolic cyt-c and Smac. As the loading control, β-actin was used.
