A BAX/BAK/CypD-Independent Intrinsic Death Pathway Figure 3. ER stress triggers cytochrome c redistribution and caspase-9-dependent cell death. (A) WT and BAX/BAK DKO cells were treated with 10 mg/ml Tm or left untreated (NT) in regular cell culture media or in cells pre-treated with media containing 0.5% serum for 2 h. After 8 h of treatment, DYm was determined by DIOC8(3) staining and FACS analysis. (B) BAX/BAK DKO cells were exposed to 10 mg/ml Tm in the presence of cell culture media containing 10% serum (top panel) or pre-treated with media containing 0.5% serum (Botton panel). After 10 h, cells were stained with Mitotracker orange (red), fixed and cytochrome C (Cyt C, green) distribution visualized by indirect immuofluorescence. DNA was stained with Hoechst (blue). Images were acquired by confocal microscopy and are representative of four independent experiments. Scale bar is 25 mm. (C) Quantification of the Manders colocalization coefficient MTO/CytC (fraction of mitrotracker orange signal in co-localizing with cytochrome C signal) or CytC/MTO (fraction of the cytochrome C co-localizing mitotracker orange). Student’s T-test was used to analyze statistical significance (*: p,0.05). PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e37782