Wildtype Bcl-2 protects from ceramide-induced ciPCD. (A) Activity of caspase-8 and -3 in wildtype Jurkat cells in response to TNF/CHX/zVAD inducing ciPCD or TNF/CHX as a proapoptotic stimulus. Cells were incubated with 100 ng/ml hTNF in combination with 2 μg/ml CHX and/or 50 μM zVAD-fmk for 4 h before activation of caspases -8 and -3 was determined by measuring the cleavage of fluorogenic substrates (zIETD-afc and zDEVD-afc) over 120 minutes. Prior to stimulation, cells were preincubated with 50 μM zVAD-fmk for 30 min (for stimulations with TNF/CHX/zVAD) or medium (stimulations with TNF/CHX). For positive control, caspases were activated in vitro by adding cytochrome c and dATP (Cyt c/dATP) to the cell extracts. (B) Wildtype (untransfected) Jurkat cells and Jurkat cells overexpressing pSFFV-Bcl-2 (Bcl-2 WT) were left untreated or stimulated with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk for 20 h before micrographs of the cells were taken to document their morphology. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. As representative examples, one healthy cell and one cell undergoing ciPCD with necrosis-like morphology are marked by a black or a white arrow, respectively. (C) In parallel, uptake of PI was determined by flow cytometry as a marker for loss of plasma membrane integrity (see „Materials and Methods‟). The percentage of viable cells (PI-negative, large) is indicated in the lower right quadrants of the dot plots. One representative experiment out of three performed is shown. (D) Quantification of cell viability data. The bar graphs represent the means from all three independent experiments, error bars indicate the respective standard deviations.