A BAX/BAK/CypD-Independent Intrinsic Death Pathway Figure 1. Serum withdrawal recovers the susceptibility of BAX and BAK DKO cells to ER stress-induced cell death. (A) Expression levels of BAX and BAK were determined in murine embryonic fibroblast from wild type (WT), BAX or BAK single knockout (KO) or BAX and BAK DKO (DKO) cells by Western blot. As a loading control actin levels was determined. (B) Phase contrast images of BAX/BAK DKO cells treated or not (NT) with 10 mg/ml Tm for 16 h in cells pre-exposed for 2 h to cell culture media containing 0.5% serum. (C) BAX and BAK DKO MEFs were treated with indicated concentrations of Tm, (D) Brefeldin A or (E) Thap in cells grown in regular cell culture media or pre-treated in media containing 0.5% serum for 2 h. WT cells grown in media containing 0.5% serum were monitored as control (see additional controls in Figure S1). After 24 h of treatment, cell viability was analyzed with the MTS assay. Mean and standard deviations are presented of triplicates of a representative experiment. (F) BAX and BAK DKO cells were pre-incubated for 2 h in cell culture media containing 0.5% serum or grown with regular cell culture media (10% serum), and then treated with a panel of different intrinsic death stimuli, including 10 mg/ml Tm. Untreated cells (NT) were used as control. After 24 h of treatment cell death was monitored by propidium iodide (PI) staining and FACS analysis. Results represent average and standard deviation of six independent experiments. Student’s T-test was used to analyze statistical significance between cells treated with Tm in the presence of 10% or 0.5% serum. (***: p,0.001). (G) Alternatively, cells were analyzed as described in F and treated with 20 mM etoposide (Eto), 1 mM taxol (Tax), 10 mM nocodazole (Noc), 10 mg/ml actinomycin D (Act D) or 0.1 mM staurosporine (Sts). Results represent average and standard deviation of 4 independent experiments. Student’s T-test was used to analyze statistical significance between cells treated with these drugs in the presence of 10% or 5% of serum (*: p,0.05; ***: p,0.001). doi:10.1371/journal.pone.0037782.g001 PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e37782