A BAX/BAK/CypD-Independent Intrinsic Death Pathway (D) Quantification of the percentage of cells with redistributed cytochrome C (CytC) experiments described in C (***: p,0.001). (E) Left panel: BAX/ BAK DKO cells exposed to 10 mg/ml Tm for 10 h in the presence of cell culture media containing 10% (top panel) or 0.5% (bottom panel) serum were fixed and active caspase 9 (green) was evaluated by indirect immunofluorescence. Actin was monitored with Phalloidin-Rhodamine (red) and nuclei were stained with Hoechst (blue). Images are representative of four independent experiments. Scale bar is 25 mm. Right panel: Quantification of active caspase 3 fluorescence per cell (Arbitrary units: A.U.) and the percentage of active-caspase 3-positive cells. Data represents mean and standard deviation of four independent experiments performed in duplicates. Student’s T-test was used to calculate statistical significance (*: p,0.05; ***: p,0.001). (F) BAX and BAK DKO cells were stably transduced with a lentiviral vector expressing an shRNA against pro-caspase-9 mRNA (shCaspase-9) or a control shRNA against the luciferase mRNA (shLuc), and pro-caspase-9 protein levels were determined by Western blot. HSP90 levels were monitored as loading control. Then, the cells were treated with 10 mg/ml Tm in cells grown under normal cell culture media or pre-treated for 2 h with media containing 0.5% serum. Cell death was determined after 24 h by PI staining and FACS analysis. Data represents mean and standard deviation of three independent experiments performed in duplicates. Student’s T-test was used to analyze statistical significance between shLuc and shCaspase-9 cells treated with Tm (*: p,0.05; n.s.: no significant). (G) BAX and BAK DKO cells were pre-incubated in media containing 0.5% serum for 2 h. Then, the cells were exposed to 10 mg/ml Tm for 24 h in present or absence of 25 mM z-IETD-fmk (caspase-8 inhibitor). As positive control, DKO cells were exposed to 1 mg/ml Fas ligand (FasL) as positive control of working Z-IETD-FMK. The cell death was monitored by propidium iodide (PI) staining and FACS analysis. Results represent average and standard deviation of three independent experiments. Student’s T-test was used to analyze statistical significance (**: p,0.01; n.s.: no significant). doi:10.1371/journal.pone.0037782.g003 withdrawal (Figure S3). As positive control we monitored ovarian tissue (Figure S3) [33,34]. The Resistance of BIM and PUMA Double Deficient Cells to ER Stress-mediated Apoptosis is Reverted by Mild Serum Withdrawal BIM, PUMA and NOXA, are strongly induced at the transcriptional level in cells undergoing prolonged ER stress, and cells deficient for these genes are partially resistant to ER stress-related injuries [30,35], whereas bim and puma double deficiency drastically protects against ER stress [11]. Based on these findings, we studied the potential role of BIM and PUMA on ER stress-mediated cell death under conditions of mild serum withdrawal. After confirming the upregulation of BIM, PUMA, and NOXA mRNA levels in MEFs undergoing ER stress (Figure 4E), we then investigated the functional role of BIM and PUMA on cell death using a gene ablation approach. BIM or PUMA single knockout cells were slightly resistant to Tm treatment when compared with WT control cells (Figure 4F). In sharp contrast, BIM and PUMA DKO cells were highly resistant to mild concentrations of Tm (Figure 4F), similar to the phenotype described in BAX/BAK DKO cells. Interestingly, the protective effects of bim and puma ablation were fully reverted when cell death stimulation was performed together with mild serum withdrawal (Figure 4G). It has be been suggested that the BH3-only protein BAD has pro-apoptotic activities independent of BAX/BAK [36]. To complement our experiments, we determined the possible pro- apoptotic effects of BH3-only proteins after enforced expression in BAX/BAK DKO MEFs under conditions of serum withdrawal. Using the pMIG-GFP bicistronic retroviral system, we transiently expressed a full panel of different BH3-only proteins including tBID, BIM, PUMA, NOXA, and BAD (Figure 4H). As previously reported [7,37], the overexpression of the activator BH3-only proteins tBID, BIM and PUMA triggers cell death in WT MEFs, which was dependent on the expression of BAX and BAK, whereas overexpression of NOXA and BAD did not affect cell viability (Figures 4H, S2B and S2C). No cell death was observed after the expression of these BH3-only proteins in BAX/BAK DKO cells when serum in the cell culture was decreased to 0.5% (Figure 4G). We controlled the efficiency of retroviral transduction in all the experiments by monitoring GFP expression by FACS analysis, which was in the range of ,40–60% efficiency (Figure S3B). Taken together, these results suggest that BH3-only proteins are not involved in the occurrence of BAX/BAK-independent apoptosis in our experimental system. Assessment of the Contribution of UPR Signaling in the Death of BAX and BAK DKO Cells Based on the observation that mild serum withdrawal specif- ically restores the susceptibility of BAX/BAK DKO cells to ER stress-mediated apoptosis and not other intrinsic death stimuli (Figure 1G), we rationalized that signals emerging from the ER may regulate the engagement of mitochondrial-mediated apopto- sis. To test this hypothesis we first analyzed the kinetics and intensity of early UPR signaling in WT and BAX/BAK DKO cells. Cells were exposed to Tm in the presence or absence of serum withdrawal, and the expression of major proximal UPR transcription factors, XBP-1 and ATF4, or the downstream transcription factor CHOP/GADD153 [24,25], were followed over time by Western blot analysis. We did not observe any significant alteration of the kinetic of UPR signaling in DKO cells treated with 10 mg/ml Tm in the presence or absence of serum withdrawal (Figure 5A). This result is in agreement with our recent finding indicating that the regulation of XBP-1 expression by BAX and BAK or BH3-only proteins is lost when cells are treated with high Tm concentrations [38,39]. More importantly, these results indicate that the intensity of ER stress is not altered by the serum withdrawal condition as monitored by the kinetics and amplitude of UPR signaling. Activation of the UPR stress sensor IRE1a has been linked to ER stress-induced apoptosis through engagement of the JNK pathway [40,41]. Treatment of cells with the selective JNK inhibitor SP600125 did not attenuate the rate of apoptosis of DKO cells exposed to Thap or Tm together with serum withdrawal (Figure 5B). Another death kinase, Glycogen Synthase Kinase-3 beta (GSK3-b), regulates ER stress-mediated apoptosis under certain conditions [42,43]. Pre-treatments with two different GSK3-b inhibitors (TDZD-8 and L803-mts) did not affect the survival of BAX and BAK DKO cells undergoing ER stress in conjunction with mild serum withdrawal (Figure 5B). Sustained activation of the ER stress sensor PERK triggers apoptosis [26,44–47], whereas early signaling events promote cell protection [48,49]. We knocked down PERK expression with shRNA and assessed the impact on cell death. An efficient down regulation of PERK was observed, reflecting an attenuation of PERK phosphorylation and the expression of ATF4 and CHOP/ GADD153 (Figure 5C). Cell death induced by Tm treatment combined with serum withdrawal was significantly enhanced by targeting PERK expression (Figure 5D). This result suggest that UPR signaling modulates in part the occurrence of BAX and BAK-independent cell death after stimulation of ER stress under conditions of mild serum withdrawal. PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e37782