A BAX/BAK/CypD-Independent Intrinsic Death Pathway Figure 6. Cell death of BAX and BAK DKO cells is independent of CypD expression. (A) Right panel: BAX/BAK DKO cells stably transfected with a SERCA expression vector or an empty vector were treated with 10 mg/ml Tm or left untreated (NT) in cells pre-incubated for 2 h with cell culture media containing 0.5% serum. After 24 h, cell viability was analyzed by PI staining and FACS analysis. Left panel: SERCA expression levels were determined by Western blot analysis in the two cell lines presented. As loading control HSP90 levels were determined. Data is representative of two independent experiments. Mean is presented. (B) 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), and the mRNA levels of cypD were determined by real time PCR and normalized by the levels of beta actin (left panel). Then, in these cells, cell viability was analyzed after treatment with 10 mg/ml Tm 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 (right panel). Mean and standard deviation is presented. p value for Tm treatment was calculated with Student’s t-test in a total of three independent experiments for indicated comparisons. n.s.: non significant (C) Confirmation of the genotype of BAX/BAK/CypD triple knockout (TKO) cells. Upper panel: The levels of BAX and BAK proteins were monitored by Western blot in TKO and WT control PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e37782