A BAX/BAK/CypD-Independent Intrinsic Death Pathway Figure 7. Working model: A BAX/BAK and CypD-independent intrinsic apoptosis pathway. ER stress-induced apoptosis is mediated by the activation of BAX and BAK at the mitochondria through upregulation of the upstream BH3-only proteins (i.e. BIM and PUMA) leading to mitochondrial outer membrane permeabilization, cytochrome c release, and subsequent apoptosome assembling. Under certain conditions such as mitochondrial calcium overload and oxidative stress, cytochrome c release can occur independently of BAX and BAK through opening of the mitochondrial permeability transition pore (PTP), which is form by several components including CypD and VDAC. Opening of the PTP leads to the expansion of the mitochondrial matrix, resulting in sufficient swelling to rupture the outer mitochondrial membrane and cytochrome c release. Stimulation of ER stress in conjunction with mild serum withdrawal triggers apoptosis in a BAX/BAK and CypD-independent manner. This alternative death pathway is mediated by the activation of caspase-9. Under normal conditions growth factor signaling may inhibit this alternative cell death pathway at the level of (i) mitochondria, or by (ii) blocking the activation of a specific ER-dependent event. doi:10.1371/journal.pone.0037782.g007 SDS-PAGE and Western Blot Analysis Cells were homogenized by sonication on ice in RIPA buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% DOC, 0.5% triton X-100) containing a protease inhibitor cocktail (Roche, Basel, Germany), NaF 1 mM, sodium orthovanadate and a phosphatase inhibitor cocktail (Promega). Protein concentration was determined by micro-BCA assay (Pierce, Rockford, IL). The equivalent of 30– 50 mg of total protein was generally loaded onto 10% or 4–12% SDS- PAGE minigels (Cambrex) and analyzed by Western blotting. The following antibodies and dilutions were used: anti-CHOP/ GADD153 1:2000, anti-ATF4 1:2000; anti-XBP-1 1:2000; anti- JNK1 1:5000 (Santa Cruz); anti-caspase-12 1:3000 (Exalpha Biologicals), anti-BAX and anti-BAK 1:1000 (Upstate Technology); anti-HA 1:2000 (Roche), anti-caspase-9 1:1000 (Pharmigen), anti- actin 1:5000 (Chemicon Inc.), anti-SERCA 1:1000 (Affinity Bior- eagents), anti-HSP90 1:1000 (Santa Cruz Biotechnology) and anti- pPERK, and p-JNK 1:2000 (Cell Signaling). After incubation with the primary antibody overnight at 4uC, membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase- coupled secondary antibodies (Amersham Biosciences) diluted 1:5000 in washing buffer. After washing, specifically bound antibodies were detected by enhanced chemiluminescence assay (Amersham Biosciences). Cytochrome c Release, Caspase Activation and Mitochondrial Membrane Potential Determination WT, and DKO cells were washed 3 times with cold PBS and then fixed for 30 min with 4% formaldehyde on ice. Immunoflu- orescence to detect cytochrome c was performed with methods previously described [63] using an anti-cytocrome c antibody (Pharmingen) and fluorescent staining. Then cells were maintained in PBS containing 0.4% formaldehyde for visualization on a confocal microscope and analysis with the IP lab v 4.04 software (Beckon and Dickenson). In cells treated with ER stressors the mitochondrial membrane potential and cellular volume was analyzed by flow cytometry (FACS; Becton Dickinson, Mountain View, CA) after staining of the cells with 2 nM 3,3-dihexylox- acarbocyanine [DiOC6(3)] (Molecular Probes) [63]. For cleaved caspase-3 and cleaved caspase-9 immunofluorescence, anti-body were used 1:250 (#95095 and #96615, Cell signaling). For mitochondrial and cytochrome C (1:250, BD Pharmigen) colocalization determinations one focal plane was analyzed. The images obtained were deconvolved and the background was subtracted. Colocalization between organelles was quantified using the Manders algorithm, as previously described [64–66]. Electron Microscopy Analysis Cells were fixed in 2.5% glutaraldehyde, 0.01% picric acid, 0.1 M cacodylate buffer (pH 7.4) for 1 h, rinsed in the same buffer and incubated in 1% OsO4 for 30 minutes followed by incubation with 2% uranyl acetate. Cells were dehydrated with a graded series of ethanol, and infiltrated with Epon (Ted Pella Inc., #18005). Ultrathin sections were contrasted with 1% uranyl acetate and lead citrate. Grids were examined with a Philips Tecnai 12 electron microscope operated at 80 kV. Negative films were developed and scanned. PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e37782