For quantification of mitochondrial morphology, photoshop software was used to quantify mitochondrial area, perimeter, circularity and mean gray value in differents treatments as we recently described [50]. RNA Extraction and RT-PCR Total RNA was prepared from cells using trizol (Invitrogen, Carlsbad, CA) and cDNA was synthesized with SuperScript III (Invitrogen, Carlsbad, CA) using random primers p(dN)6 (Roche, Basel, Switzerland). Quantitative real-time PCR reactions employ- ing SYBR green fluorescent reagent were performed in an ABI PRISM 7700 system (Applied Biosystems, Foster City, CA). The relative amounts of mRNAs were calculated from the values of comparative threshold cycle by using b-actin as control. Primer sequences were designed by Primer Express software (Applied Biosystems, Foster City, CA) or obtained from the Primer Data bank (http://pga.mgh.harvard.edu/primerbank/index.html). Real time PCR was performed as previously described [38] using the following primers: cyclophilin D 59-GGGACAGGTGGCGAAAGTATT-39 and 59-TTCGTATTGGGGCCTGCATTT-39; bim 59-CGA- CAGTCTCAGGAGGAACC-39 and 59-CATTTGCAAA- CACCCTCCTT-39; puma 59-GCCCAGCAGCACTTAGAGTC- 39 and 59-GGTGTCGATGCTGCTCTTCT-39; noxa 59- GGAGTGCACCGGACATAACT-39 and 59-TTGAGCA- CACTCGTCCTTCA-39; caspases-9, 59-TCCTGGTACATCGA- GACCTTG-39and 59-AAGTCCCTTTCGCAGAAACAG-39, ac- tin 59-TACCACCATGTACCCAGGCA-39 and 5- ‘CTCAGGAGGAGCAATGATCTTGAT-39. Supporting Information Figure S1 Serum withdrawal recovers the susceptibility of BAX and BAK DKO cells to ER stress-induced cell death. WT MEFs were treated with 10 mg/ml Tm, 10 mM Thap or 20 mM brefeldin A in cells grown in regular cell culture media or in media containing 0.5% serum. Mild serum withdrawal was performed 2 h before the addition of ER stress agents. After 24 h of treatment, cell viability was analyzed with the MTS assay. Results are representative of two independent experiments performed in triplicate. Mean and standard devia- tions are presented of a representative experiment. (TIF) Figure S2 BCL-XL in the control of cytocrome c redistribution and BH3-only protein expression and efficiency of retroviral transduction. BAX/BAK DKO MEFs that stably transduced with retroviruses expressing human BCL-XL or empty vector (Mock) cells were exposed to 20 mg/ml Tm in the presence of cell culture media containing 10% serum or pre-treated with media containing 0.5% serum. After 10 h, cells were stained with Mitotracker orange (red), fixed and cytochrome C (Cyt C, green) distribution visualized by indirect immuofluor- escence. Images were acquired by confocal microscopy. Then, quantification of the Manders colocalization coefficient MTO/ CytC (fraction of mitrotracker orange signal in co-localizing with cytochrome C signal) or CytC/MTO (fraction of the cytochrome C co-localizing mitotracker orange) were analyzed. Data represent the average and standard deviation of four independent experiments. One-way ANOVA test was used to analyze statistical significance (*: p,0.05; **: p,0.01; ***: p,0.01). (B) The expression of the HA-tagged BH3-only proteins was analyzed by Western blot after transfection of 293T packaging cells with pMIG-GFP retroviral vectors together with packaging vector to produce retroviruses employed in Figure 4F. Red arrowheads A BAX/BAK/CypD-Independent Intrinsic Death Pathway indicate the protein bands corresponding to each BH3-only protein. A HA-BAX expression vector was also used as control. (B) The percentage of retroviral transduction efficiency was assessed in WT and BAX/BAK DKO cells by monitoring the bicistronic expression of GFP by FACS analysis. Results are representative of two independent experiments performed in duplicated. Means are presented of a representative experiment. (TIF) Figure S3 Low mRNA levels of pro-apoptotic protein BOK in WT and BAX/BAK DKO cells. WT and BAX/BAK DKO cells were treated with 10 mg/ml Tm or left untreated. DKO cells were also pre-incubated for 2 h in cell culture media containing 0.5% serum. After 6 h of treatment, bok mRNA levels were assessed by real time PCR. As control, the levels of bok mRNA expression were also monitored in mRNA extracts from mouse ovary. (TIF) Figure S4 Cell death in BAX and BAK DKO cells is independent of CypD. (A) BAX/BAK DKO cells were stably transduced with lentiviral vectors expressing an shRNA against cypD mRNA (shCypD) or control shRNA against the luciferase mRNA (shLuc). Then, cell viability was analyzed after treatment with 10 mM Thap in cells grown in regular cell culture conditions or pre-incubated for 2 h in media containing 0.5% serum. Untreated cells (NT) are also presented. After 24 h cell death was monitored by PI staining and FACS analysis. (B) WT and BAX/BAK/CypD TKO cells were treated with 10 mM Thap under regular cell growing conditions or in cells pre-treated with media containing 0.5% serum for 2 h. After 24 h, cell death was analyzed by PI staining and FACS analysis. Mean and standard deviation are presented of three determinations. (TIF) Figure S5 Effect of Cyclosporine A in cell death in WT and BAX and BAK DKO cells exposed to ER stress. (A) BAX/BAK WT cells were or not pre-treated with CsA 10 mM for 30 min. Then, cells were incubated with 0.25, 1 and 2,5 mg/mL Tm for 24 h. Cell viability was analyzed by PI staining and FACS analysis. Data is representative of three independent experiments. Mean and standard deviation are presented. Significant differenc- es were obtained between indicated control and experimental groups using Student’s t-test (** p,0.01). (B) BAX/BAK DKO cells were pre-treated with cell culture media containing 0.5% serum for 2 h in the presence or absence of 1 mM of CsA. Then, cells were exposed to Tm or Thap for 24 h. Cell viability was analyzed by PI staining and FACS analysis. Data is representative of two independent experiments. Mean is presented. (TIF) Acknowledgments We thank Dr. Fabio Martino, Dr. Diego Rodriguez, and also members of the Korsmeyer’s lab for helpful discussion. We would like to thank Dr. Y. Ysujumoto for providing BAX/BAK/CypD TKO and WT, cells and Dr. Nir Hacohen and The Broad Institute (Boston, USA) for providing shRNA lentiviral constructs. We thank Monica Perez for excellent EM processing. Author Contributions Conceived and designed the experiments: SZ DRR FC VP SL FL RV EC SK CH. Performed the experiments: SZ DRR FC VP FL RV. Analyzed the data: SZ DRR FC VP FL RVEC SKCH. Contributed reagents/materials/analysis tools: FC EC SK SL CH. Wrote the paper: SZ DRR CH. PLoS ONE | www.plosone.org 14 June 2012 | Volume 7 | Issue 6 | e37782