A BAX/BAK/CypD-Independent Intrinsic Death Pathway between MOCK and BCL-XL cells (***: p,0.001). (C) Cells described in A were treated with 20 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 distribution visualized by indirect immunofluorescence (Cyt C, green). DNA was stained with Hoechst (blue). Images were acquired by confocal microscopy and are representative of four independent experiments. Scale bar is 25 mm. (D) Quantification of the percentage of cells with redistributed cytochrome C (CytC) experiments described in C (**: p,0.01). (E) The mRNA levels of bim, puma and noxa were analyzed by semi-quantitative RT-PCR in total cDNA from MEFS treated with 10 mg/ml Tm for indicated time points. Beta-actin levels were determined as loading control. (E) WT, BIM KO, PUMA KO, and BIM and PUMA double deficient (DKO) cells were treated with 0.5 mg/ml Tm for 24 h, and then cell death was evaluated after PI staining and FACS analysis. Data represents two independent experiments performed in triplicates. Mean and standard deviation is presented of a representative experiment. (F) BIM and PUMA DKO cells and parental WT controls cells were treated with indicated concentrations of Tm as described in (E). After 24 h, apoptosis was determined by quantifying cells with hypo-diploid DNA content after PI staining of permeabilized cells and FACS analysis. Mean and standard deviation is presented. (G) WT and BAX/BAK DKO cells were transduced with pMIG-GFP retroviruses expressing tBID-HA, BIM-HA, PUMA-HA, NOXA-HA, BAD-HA, or empty vector (MOCK). After 8 h of viral transduction, serum in the culture media was decreased to 0.5% or replaced by media containing 10% serum. Then, cell viability was analyzed after 24 h by PI staining and FACS analysis. Transduction efficiency in each experiment was monitored by detecting GFP expression by FACS in the same experiments (example in Figure S2). Data is the average and standard deviation of three independent experiments. p value to compare the rate of cell death in WT cells transduced with BH3-only protein-expressing viruses versus a MOCK transduced WT cells were calculated by Student’s t-test (*: p,0.05; **: p,0.001; n.s.: non significant). Comparison between DKO cells transduced with retroviruses in the presence or absence of serum were in all cases non significant. doi:10.1371/journal.pone.0037782.g004 Figure 5. UPR signaling in BAX/BAK DKO cells undergoing ER stress and mild serum withdrawal conditions. (A) WT and BAX/BAK DKO cells were treated with 10 mg/ml Tm under regular growing conditions or in cells pre-incubated for 2 h with media containing 0.5% serum. After indicated time points, the expression levels of the UPR transcription factors XBP-1, ATF4 and CHOP were assessed by Western blot analysis. As control, the levels of HSP90 were also determined. (B) BAX/BAK DKO cells were pre-treated with 1 mM of the JNK inhibitor SP600125 or 10 mM two different GSK3-b inhibitors (TDZD-8 and L803-mts) for 30 min and then treated with 10 mg/ml Tm or 10 mM Thap under regular growing conditions or in cells pre-treated for 2 h with media containing 0.5% serum. After 16 h, Annexin-V-FITC staining was evaluated using FACS analysis. Data represents mean and standard deviation of three determinations. (C) WT and BAX/BAK DKO cells were stably transduced with a pool of lentiviral vectors expressing different shRNA constructs against Perk mRNA (shPERK) or control shRNA against the luciferase mRNA (shLuc). To determine the efficiency of knockdown, the level of phospho-PERK, and the downstream targets ATF4 and CHOP were assessed by Western blot in untreated cells (NT) or treated with 10 mg/ml Tm for 4 h. (D) Cells described in (C) were treated with Tm as described in (B) and cell death was monitored after 20 h by PI staining and FACS analysis. In addition, WT control cells were stably transduced with shPERK and shLuc lentiviral constructs for comparison in the same experiments. Data represents the mean and standard deviation of four independent experiments. p values were calculated with Student’s t-test (***: p,0.001, **: p,0.01 n.s.: non significant). doi:10.1371/journal.pone.0037782.g005 PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e37782