A BAX/BAK/CypD-Independent Intrinsic Death Pathway cells. HSP90 levels were used as loading control. Bottom panel: The cypD genotype was assessed by PCR using genomic DNA of WT or TKO cells. PCR fragments corresponding to WT and cypD KO alleles are indicated. (D) Right panel: BAX/BAK/CypD TKO and parental control (WT) cells were analyzed as described in (B). Data represents mean and standard deviation of six independent experiments performed in duplicates. p value was calculated with Student’s t-test for indicated comparisons. n.s.: non significant. Left panel: phase contrast images to visualize morphological changes of TKO cells pre-incubated with cell culture media with 0.5% serum treated or not (NT) with 10 mg/ml Tm. (E) Upper panels: Cell morphology was monitored in BAX/BAK/CypD TKO cells treated as described in (D) using EM analysis. Arrowheads indicate classical apoptosis morphological features including chromatin condensation and fragmentations. In addition, cell shrinkage is observed in the analysis. N: nucleus. Lower panels: In the same cells presented in the upper panel, mitochondrial (M) morphology was visualized. A representative image is presented. (F) The area and circularity of mitochondria were determined in TKO cells in cells described in (c). Mean and standard deviation is presented of the analysis of 78 (0.5% serum), 105 (Tm) and 112 (Tm +0.5% serum) individual mitochondria per group respectively. (G) TKO cells were pre-treated with 100 mg/ml AEBSF, 1 mM Trolox or 5 mM N-acetylcysteine (NAC), for 30 min and then treated with 10 mg/ml Tm under regular growing conditions or in cells pre-treated for 2 h with media containing 0.5% serum. After 16 h, PI staining was evaluated using FACS analysis. (H) As positive control for the use of AEBSF, BAX and BAK DKO cells were treated with 100 mM arachidonic acid together with 10 mM A23187 in the presence or absence of 100 mg/ml AEBSF, and then after 16 h cell viability analyzed by PI staining and FACS analysis. doi:10.1371/journal.pone.0037782.g006 with complete serum withdrawal. This phenomenon occurred only in a very small population of cells and with a slow kinetic of cytochrome c release [58], that is mediated by CypD [59]. We excluded the participation of this pathway in our experimental system since we were not able to protect BAX/BAK DKO cells against Tm/serum withdrawal-induced cell death after stimulating cells with insulin or IGF1 or treating with Phosphoinositide 3- kinases (PI3Ks) inhibitors (not shown). It remains to be determined how growth factor signaling blocks BAX/BAK/CypD-indepen- dent apoptosis. This inhibitory activity may operate at the level of the mitochondria to trigger cytochrome c release or it may block a yet unknown death-signaling event at the level of the ER (Figure 7). We are currently investigating additional models to uncover the molecular identity of this intrinsic BAX/BAK/CypD-independent death pathway. The possible involvement of lipids such as ceramides in the process constitute an interesting model since they form large lipid ion-channels in vitro [60] and can also induce the release of cytochrome c from purified mitochondria [61]. Moreover, ceramides and related lipids could mediate the crosstalk between ER and mitochondria in the induction of apoptosis in certain experimental systems [62]. The occurrence of BCL-2 family-independent and CypD-independent apoptosis may be taken into consideration when therapeutic strategies are designed to target this group of apoptosis regulators in a disease context, since depending on the cellular environment (i.e. presence of particular growth factors), and the cross-talk between different death signals, the way how apoptosis is regulated may drastically differ from our classical view of the process. Materials and Methods Materials Tunicamycin (Tm), brefeldin A, thapsigargin, zVAD-fmk, zIETD-fmk, GSK3 inhibitors and Ruthenium red were purchased from Calbiochem EMB Bioscience Inc. IGF-1 was purchased from Australbiologicals and insulin from Actrapid. Cyclosporin A, etoposide, taxol, PI3K inhibitors, JNK inhibitors, TDZD-8 and L803-mts, TNF-a and PD98059 were obtained from Calbiochem. Cell medium, fetal calf serum and antibiotics were obtained from Life Technologies (Maryland, USA). Hoechst, 3,3-dihexyloxacar- bocyanine [DIOC6(3)] and fluorescent secondary antibodies were purchased from Molecular Probes. Nocododazole, actinomycin D were obtained from Sigma. AEBSF was obtained from Roche. Cell Culture, Detection of Cell Death and Apoptosis SV40 transformed MEFs were generated and cultured as described [29]. BAX and BAK DKO cells expressing SERCA or mitochondrial targeted BAX were previously described [27]. BAX/ BAK/cyclophilin D triple knockout MEFs were previously described [22] and were kindly provided by Dr. Tsujimoto. Apoptosis was determined by FACS analysis of cells stained with Annexin-V FITC and Propidium Iodide (Promega) as previously described [29]. Analysis was performed using the Cell Quest program. Hypodiploid cells were quantified by FACS as previously described [63]. In parallel, nuclear morphology was analyzed by Hoechst33342 staining (Molecular Probes). To address the impact of serum withdrawal on ER stress-induced apoptosis, before treatment with ER stress agents, cells were pre-incubated with cell culture media containing 0.5% serum (fetal calf serum) for 2 h and then treated with brefeldin A, tunicamycin or thapsigargin. For caspase inhibition, cells were pre-treated with 10 mM zVAD-fmk for 30 min. Alternatively, cell viability was quantified in 96-well plates using 3-(4,5- dimethylthazol-2-yl)-5-3-carboxymethoxy-phenyl)-2-(4-sulfophe- nyl)-2H-tetrazolium (MTS) and phenazine methosulfate according to the recommendations of the supplier (CellTiter 96 AQueous; Promega, Madison, WI). Mitochondria targeted HA-BAX (mtBAX) was constructed by fusing the 29 amino acid mitochondrial targeted sequence from subunit VIII of human cytochrome c oxidase (COX VIII) into the N-terminal end of HA-BAX and was present on the OMM surface [27]. Retroviral expression vectors for HA-tagged BH3-only proteins were previously described [7]. The production of amphitropic retroviruses using the 293GPG packing cell line was performed as described previously [29]. Retroviral plasmids were transfected using Effectene (Qiagene) according to the manufactur- er’s protocols. Retroviral transduction of each indicated BCL-2 family protein was confirmed by Western blotting using an anti-HA antibody. Knockdown of CypD, Caspases-9 and PERK We generated stable MEFs with reduced levels of cypD, caspases- 9 and perk mRNA using methods previously described [38] by targeting each mRNA with shRNA using the lentiviral expression vector pLKO.1 and puromycin selection. As control a shRNA against the luciferase gene was employed. Constructs were generated by The Broad Institute (Boston, USA) based on different criteria for shRNA design (see http://www.broad.mit. edu/genome_bio/trc/rnai.html). For each gene we screened a total of five different constructs and selected the most efficient using real time PCR analysis. We identified the following targeting sequences for caspase-9 (CCTTTGTTCATCTCCTGCTTA) and cypD (GCAGAATTGCTTAAAGTCAAA). For knocking down PERK a mix of five constructs was employed since none of the five targeting sequences alone were efficient to down regulate PERK. Targeting sequences are CCTCTACTGTTCACTCAGAAA, CCATGAGTTCATCTGGAACAA, GCCTGTTTGATGATA- CAAGTT, CCATACGATAACGGTTACTAT and GCCACTTTGAACTTCGGTATA. PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e37782