A BAX/BAK/CypD-Independent Intrinsic Death Pathway Figure 2. BAX and BAK double deficient cells undergo caspase-dependent apoptosis under conditions of ER stress in conjunction with serum withdrawal. (A) Left panel: WT, BAX/BAK DKO cells or DKO cells stably reconstituted with mitochondrial-targeted BAX (mtBAX) were treated with 10 mM Thap or 10 mg/ml Tm under normal growing conditions (10% serum). After 12 h, apoptosis was monitored by Annexin-V-FITC and PI staining followed by FACS analysis. Mean and standard deviations are presented of three determinations. Right panel: Alternatively, experiments were performed in cells pre-treated for 2 h with cell culture media containing 0.5% serum, followed by the addition of ER stress agents. (B) BAX/BAK DKO cells were exposed to 10 mM Thap in the presence of cell culture media containing 10% serum or pretreated in media containing 0.5% serum, and then cells were stained with Hoechst to visualize nuclear morphology by fluorescent microscopy. (C) Cell morphology was monitored in BAX/ BAK DKO cells treated with Tm for 12 h or 16 h under normal growing conditions or in cells pretreated for 2 h with media containing 0.5% serum. Untreated (NT) cells are also presented as control. Red arrowheads indicate classical apoptosis morphological features including chromatin PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e37782