A BAX/BAK/CypD-Independent Intrinsic Death Pathway condensation and fragmentations. In addition cell shrinkage is observed in the analysis. N: nucleus. (D) BAX/BAK DKO cells were exposed to 10 mg/ml Tm in the presence of cell culture media containing 10% serum (top panel) or pretreated in media containing 0.5% serum (Botton panel). After 10 h, active caspase 3 (green) was evaluated by indirect immunofluorescence. To visualize cells, actin was monitored with Phalloidin-Rhodamine staining, and nuclei were stained with Hoechst (blue). Images were acquired with a confocal microscope and represent the result of four independent experiments. Left panel: Quantification of the levels of active caspase-3 per cell by measuring fluorescent pixel intensity/area (A.U.) or the percentage of cells with positive active caspase-3 signal over a cutoff definer in non treated cells. Mean and standard deviation is presented of four experiments. Student’s T-test was used to analyze statistical significance between both groups (*: p,0.05; ***: p,0.001). (E) BAX/BAK DKO cells were pretreated for 30 min with 10 mM zVAD-fmk or left untreated in media containing 10% or 0.5% serum (pre-incubation of 2 h), and then exposed to 10 mg/ml Tm or 10 mM Thap. After 12 h, Annexin-V-FITC staining was evaluated by FACS analysis. Mean and standard deviation is presented of three determinations. (F) In parallel, the correlation between Annexin-V-FITC staining and cell shrinkage was evaluated by FACS by monitoring forward scatter parameter in cells described in (E). Data represent the results of three independent experiments. doi:10.1371/journal.pone.0037782.g002 We confirmed the appearance of apoptosis markers in BAX/ BAK DKO cells treated with Tm under mild serum withdrawal by the visualization of chromatin condensation and fragmentation by nuclear staining with Hoechst (Figure 2B). We also detected ultrastructural features of apoptosis under our experimental conditions using electron microscopy (EM), associated with the appearance of condensed and fragmented nuclei, in addition to cell shrinkage (Figure 2C). Consistent with these results, a strong activation of caspase-3 was detected using immunofluorescence when DKO cells were co-stimulated with Tm and low serum concentrations (Figure 2D). Minimal activation of caspase-3 was detected in DKO cells treated with Tm under normal growing conditions (Figure 2D). In order to test the possible involvement of caspases in the death of BAX and BAK DKO cells, we pre-incubated cells with the pan- caspase inhibitor zVAD-fmk for 30 minutes before treatment with Tm or Thap in the presence of 0.5% serum in the cell culture media. zVAD-fmk treatment drastically reduced the levels of Annexin-V-FITC staining and cell shrinkage observed in DKO cells (Figure 2E). Cell shrinkage was also observed by FACS analysis of DKO cells reflected on a decrease in the forward side scattering (FSC) parameter, which directly correlated with the levels of phosphatidylserine exposure at the cell surface (Figure 2F). Thus, mild serum withdrawal fully reverts the resistance of BAX and BAK DKO cells to ER stress-mediated apoptosis. Cell Death in BAX and BAK DKO Cells is Associated with Cytochrome c Release, a Drop in DYm, and is Mediated by Caspase-9 To define the potential involvement of mitochondria in the occurrence of BAX/BAK-independent apoptosis, we first mea- sured DYm in cells undergoing ER stress. As expected, whereas WT MEFs presented a clear drop on DYm after Tm treatment, BAX/BAK DKO cells showed no decrease on DYm as determined by DIOC8(3) staining and FACS analysis (Figure 3A). This phenotype was partially reverted by performing treatments with Tm in conjunction with serum withdrawal (Figure 3A). To further examine the contribution of mitochondria in the cell death process, we visualized the distribution of cytochrome c in BAX/ BAK DKO cells by immunofluorescence. As shown in Figure 3B, cytochrome c redistribution was observed in DKO cells treated with Tm in conjunction with serum withdrawal, resulting in the partial loss of the punctuate pattern characteristic of mitochon- dria-located cytochrome c into a more diffuse cytoplasmic pattern (Figure 3B). Quantification of four independent experiments revealed a significant reduction in the Manders co-localization index between cytochrome c and a mitochondrial tracker when ER stress was stimulated in conjunction with mild serum withdrawal (Figure 3C). Moreover, at the population level, around 60% of BAX/BAK DKO cells presented cytochrome c redistri- bution using this imaging analysis after ER stress and serum withdrawal stimulation (Figure 3D). We then monitored the possible assembly of the apoptosome by monitoring the activation of caspase-9 using immunofluorescence using a specific antibody against active caspase-9. A consistent activation of caspase-9 was detected in most BAX and BAK DKO cells when treated with Tm and low serum concentration in terms of signal intensity or percentage of positive cells (Figure 3E). To define the functional contribution of the canonical mitochondrial- apoptotic pathway in the death of BAX/BAK DKO cells, we knocked down the expression of pro-caspase-9 using stable delivery of a short hairpin RNA (shRNA) with lentiviral vectors, which only partially reduced pro-caspase-9 levels (Figure 3F). As control, an shRNA construct against luciferase mRNA was employed. Knocking down pro-caspase-9 expression significantly decreased the rate of cell death observed in BAX/BAK DKO cells treated with Tm in conjunction with serum withdrawal (Figure 3F, right panel). As additional control, we also tested the possible contribution of caspase-8 to the death process of BAX/BAK DKO cells. Cells were pre-incubated with 40 mM of the caspase-8 inhibitor z-IETD-fmk for 30 min, and the levels of cell death were evaluated by FACS. z-IETD-fmk did not significantly reduce ER stress-induced apoptosis under conditions of mild serum with- drawal (Figure 3G). In contrast, z-IETD-fmk efficiently blocked FasL-induced apoptosis in BAX/BAK DKO cells (Figure 3G). Together, these results suggest an active role of the apoptosome in the death of BAX and BAK DKO cells after stimulation of ER stress in the presence of mild serum withdrawal. Cell Death in BAX and BAK DKO Cells is Insensitive to BCL-XL Overexpression Anti-apoptotic BCL-2 family members are potent inhibitors of intrinsic cell death by antagonizing BH3-only proteins. We stably transduced BAX and BAK WT and DKO cells with retroviruses expressing BCL-XL or the empty vector (Figure 4A) and then assessed their susceptibility to cell death after ER stress/mild serum withdrawal stimulation. As expected, BCL-XL overexpres- sion dramatically protected WT cells against ER stress-mediated apoptosis (Figure 4B, left panel). However, no reduction in the amount of cell death was observed in BAX/BAK DKO cells overexpressing BCL-XL after 24 h of treatment with Tm in conjunction with serum withdrawal (Figure 4B). Similarly, time course analysis revealed a similar kinetic of cell death in BAX and BAK DKO control and BCL-XL overexpressing cells (Figure 4B). In agreement with these results, BCL- XL overexpression did not alter the release of cytochrome c in BAX/BAK DKO cells exposed to similar conditions (Figure 4C), which was confirmed after quantification and statistical analysis (Figures 4D and S2A). In addition to BAX and BAK, the pro-apoptotic multidomain protein, BCL-2 Ovarian Killer (BOK), remains poorly character- ized [32]. We monitored the levels of bok mRNA in WT and BAX/BAK DKO MEFs by quantitative RT-PCR. A very low abundance of bok mRNA was observed at basal levels in MEFs, which was not altered under conditions of ER stress and serum PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e37782