PTP is observed under conditions of mitochondrial calcium overload, especially when accompanied by oxidative stress, elevated phosphate concentrations and adenine nucleotide deple- tion, enabling free passage into the mitochondria of molecules of ,1.5 kDa [15–17]. Opening of the PTP leads to dissipation of the mitochondrial transmembrane potential (DYm) and an influx of solutes. This causes expansion of the matrix, resulting in sufficient swelling to rupture the outer mitochondrial membrane and cytochrome c release. However, dissipation of DYm can also lead to a sudden decrease in ATP levels, triggering necrotic cell death. Although the molecular identity of PTP remains uncertain, different components are proposed including voltage-dependent anion channel (VDAC), the adenine nucleotide translocator, the mitochondrial phosphate carrier, and Cyclophilin D (CypD), a cyclosporin A target [15–17]. Studies using knockout cells for putative components of the PTP confirmed only a functional role for CypD in PTP-mediated cell death in vitro and in vivo as we and other described [18–21]. Remarkably, physical interactions between BCL-2 family members and components of the PTP are also reported, suggesting that BCL-2-related proteins may facilitate PTP under certain conditions, possibly forming mixed protein complexes with membrane permeabilizing activities (reviewed in [17]). Interestingly, Y. Tsujimoto’s group reported the engagement of apoptosome dependent apoptosis that was independent of the expression of BAX, BAK and CypD, a phenomena initiated when death stimulation was induced by the simultaneous exposure of cells arachidonic acid and the ionophore A23187 [22], stimuli known to alter endoplasmic reticulum (ER) calcium homeostasis among other effects. Cell death after arachidonic acid and A23187 treatment was dependent on the activity of an unknown serine protease. In addition to operate as a major calcium reservoir of the cell, the ER serves as a specialized compartment for protein folding along the secretory pathway. A number of conditions interfere with protein folding at the ER lumen leading to the accumulation of unfolded or misfolded proteins, a cellular condition referred to as ‘‘ER stress’’ [23]. Cells adapt to ER stress through the engagement of the Unfolded Protein Response (UPR), an integrated signaling pathway that controls protein folding at the ER [24,25]. Under chronic ER stress conditions, cells undergo apoptosis mediated by the activation of BAX and BAK at the mitochondria [26]. BAX and BAK double deficient (DKO) cells [3,27,28] and conditional DKO mice [29] are highly resistant to death stimuli dependent on ER injuries. ER stress-induced apoptosis is initiated by the activation of several BH3-only protein including BIM, PUMA and NOXA, and is antagonized by the expression of BCL- 2 or BCL-XL [10,30,31]. In this article, we provide evidence indicating the occurrence of cytochrome c release and caspase-9- dependent apoptosis in BAX and BAK DKO cells when ER stress is stimulated in combination with mild serum withdrawal. Interestingly, BAX and BAK-independent cell death did not require CypD expression, did not involve mitochondrial swelling, and was insensitive to serine protease inhibitors. Our results are indicative of the existence of an alternative intrinsic cell death pathway that is independent of BAX/BAK and CypD expression that possibly triggers apoptosome-mediated cell death. Results Mild Serum Withdrawal Recovers the Susceptibility of BAX and BAK DKO Cells to ER Stress-induced Cell Death, but Not to other Intrinsic Death Stimuli We previously reported an impaired ability of BAX and BAK DKO murine embryonic fibroblasts (MEFs) to activate the ER A BAX/BAK/CypD-Independent Intrinsic Death Pathway stress sensor IRE1a, associated with diminished downstream cJun-N terminal kinase (JNK) signaling [29]. To assess the impact of BAX and BAK deficiency on JNK phosphorylation in that study, we stimulated cells in the presence of low serum concentrations (0.5–2% serum) for 2 h to decrease basal phosphorylation levels, and then exposed cells with the ER stress agent tunicamycin (Tm, inhibitor of N-linked glycosyla- tion). Interestingly, when cells were cultured under these conditions for prolonged time (.16 h), we noticed morphological changes reminiscent of apoptosis in BAX and BAK DKO cells, including the appearance of blebs at the cell surface, cell shrinkage and detachment from the cell culture plate (Figure 1A and 1B. C. Hetz and S.J. Korsmeyer, 2004, unpublished observations). To further characterize the death mechanism of BAX and BAK DKO cells under combined stimulation of ER stress and mild serum withdrawal, cells were treated with a panel of drugs that perturb ER homeostasis including Tm, brefeldin A (an inhibitor of ER to Golgi trafficking), or thapsigargin (Thap, inhibitor of the ER calcium ATPase SERCA) in the presence of regular cell culture media containing 10% serum or pre-treated for 2 h with media containing 0.5% serum. After 24 h, relative cell number was monitored with the MTS assay. To our surprise, resistance of BAX and BAK DKO cells to ER stress was reverted when stimulation was performed under conditions of mild serum withdrawal (Figure 1C-E and Figure S1). Serum withdrawal alone did not induce cell death of BAX and BAK DKO cells, even after three days of treatment (not shown) as previously described [3]. Similar results were observed when cell death was monitored using propidium iodide (PI) staining and FACS analysis (Figure 1F). In order to define whether mild serum withdrawal sensitizes DKO cells to other intrinsic death stimuli, cells were treated with a variety of pro-apoptotic agents that are non-ER stress related. These include etoposide (DNA damage agent), taxol and nocodazole (both alter microtubule dynamics), and actinomycin D (an inhibitor of protein synthesis). Unexpectedly, the resistance of BAX/BAK DKO cells to each of these pro-apoptotic agents was not reverted by mild serum withdrawal (Figure 1G). Similarly, serum withdrawal did not enhance the susceptibility of BAX/BAK DKO cells to engagement of death receptors since a similar rate of cell death was observed after treatment with TNFa when compared to control cells grown in the presence of 10% serum (,80% cell death, not shown). These results suggest that combinatorial stimulation between ER stress and mild serum withdrawal engages a BAX/BAK-independent cell death path- way. Caspase-dependent Apoptosis in BAX and BAK DKO Cells Undergoing ER Stress in Conjunction with Serum Withdrawal To determine the mechanism involved in the death of BAX/ BAK DKO cells undergoing ER stress under mild serum withdrawal conditions, we performed Annexin-V-FITC and PI staining. As previously described [3,27,28], DKO cells grown in cell culture media containing 10% serum were completely resistant to apoptosis induced by ER stress (Figure 2A). Reconstitution of DKO cells with mitochondria-located BAX restored the sensitivity of these cells to Tm-mediated cell death (Figure 2A, left panel), confirming the requirement of BAX expression at the mitochon- dria to initiate apoptosis by ER-related injuries [27]. BAX and BAK DKO cells exposed to Tm or Thap in conjunction with mild serum withdrawal underwent apoptosis measured after Annexin- V-FITC staining (Figure 2A, right panel). PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e37782