Knockdown of c-FLIP restores sensitivity toward CD95-mediated apoptosis upon Tax expression. (A) Jurkat ERtax and ERΔtax cells were stimulated with 20 μg/mL anti-CD3 for 16 hours and subsequently transfected with vectors encoding c-FLIP or control (ctl) siRNA. Cells were then treated with 2 μMHT for 24 hours and viable cells were recovered by gradient centrifugation. mRNA expression levels of c-FLIP_L (top panel) and c-FLIP_S (bottom panel) were assessed by quantitative PCR and normalized to β-actin mRNA. (B) Cells were treated and transfected as described in panel A. Apoptosis was triggered with the indicated concentrations of LZ-CD95L for 16 hours and analyzed by annexin V staining of electronically gated eGFP⁺ cells. (Top panel) Data of 3 independent experiments are shown normalized with respect to apoptosis induction of ERΔtax cells transfected with control siRNA (nonnormalized data for all experiments are provided in Figure S3). Error bars represent SEM. (Bottom panel) Individual experiment showing nonnormalized data in triplicate ± SEM. c-FLIP siRNA indicates cells transfected with siRNA against c-FLIP; and ctl, cells transfected with control siRNA. Specific apoptosis was calculated as described in “Materials and methods.”