Inhibition of c-FLIP up-regulation sensitizes activated T cells to apoptosis. (A) CEM cells were either left untreated or stably transduced by lentiviral infection with a control shRNA or shRNAs targeting c-FLIP_long (FLIP_L), c-FLIP_short (FLIP_S), or both c-FLIP_long and c-FLIP_short (FLIP_L/S). Efficient knock-down of the various c-FLIP isoforms was confirmed by Western blotting. Tubulin served as a loading control. (B) Left panel: CEM cells stably transduced with the indicated shRNAs were stimulated for 14 hours with PMA and ionomycin with or without neutralizing CD95L antibody (αCD95L; 5 μg/mL, clone 5G51). Apoptotic cells were quantified by measuring DNA fragmentation. Right panel: Up-regulation of CD95L in unstimulated or PMA/ionomycin-treated CEM cells expressing the indicated shRNAs was analyzed by RT-PCR. (C) CEM cells stably transduced with the indicated shRNAs were either left untreated (top panel) or stimulated with PMA and ionomycin (bottom panel). Two hours later CD95L was added at the indicated concentrations and apoptosis was quantified after an additional 8 hours of stimulation. (D) CEM cells stably transduced with the indicated shRNAs were left untreated or stimulated for 2 hours with PMA and ionomycin in the presence or absence of 8 μM CsA. The cells were then cultured for an additional 8 hours with or without 40 ng/mL CD95L, before apoptosis was determined by flow cytometric measurement of DNA fragmentation.