Wildtype, but not organelle-restricted Bcl-2 protects stably transfected Jurkat cells from ceramide-mediated ciPCD. (A) Expression of pRc/CMV-encoded Bcl-2 WT, Bcl-2 cb5, Bcl-2 ActA and Bcl-2 ΔTM-constructs in stably transfected Jurkat cells. Cell lysates were prepared from untransfected Jurkat cells or Jurkat cells stably transfected with empty vector or with Bcl-2 constructs targeted to the ER, mitochondria, both, or the cytosol as indicated. Expression of the constructs was verified by immunoblot with a Bcl-2-specific antibody (sc-7382, Santa Cruz). Multiple bands result from detection of the endogenous Bcl-2 protein in addition to the construct (see untransfected Jurkat cells). (B) Flow cytometric analysis of PI-uptake in untransfected and stably transfected Jurkat cells. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. After that, ceramide-mediated ciPCD was induced by treatment with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk for 48 h, or the cells were left completely untreated. The percentage of viable cells is shown in the lower right quadrants of the dot plots. (C) Bar graphs showing the fraction of viable cells for each of the stably transfected lines depicted in (B). Similar results were obtained in two additional experiments with different incubation times (24 and 72 h), although with increased or reduced overall viability (data not shown).