MUC16 increases cFLIP expression to attenuate TRAIL-induced apoptosis. (A) Real-time PCR analysis of cFLIPS and cFLIPL transcript levels in Ctrl scFv and MUC16 scFv-expressing OVCAR3 cells. Results were standardized using primers of the housekeeping gene RPLPO. Data are expressed as fold change relative to levels observed in Ctrl scFv-expression OVCAR3 cells. Data are from two independent experiments. (B) Lysates from Ctrl scFv- and MUC16 scFv-expressing OVCAR3 cells were immunoblotted with cFLIPL and cFLIPS antibodies. (C) OVCAR3 Ctrl scFv and OVCAR3 MUC16 1:9#7 and 1:9#9 scFv were incubated with cycloheximide (100 μM). Lysate were obtained at different time points and immunoblot analysis was performed with anti-cFLIPL antibody. Intensity of cFLIPL signal was determined by densitometric scanning and plotted as the percentage of cFLIPL remaining compared with the control (0 h). (D) OVCAR3 Ctrl scFv and OVCAR3 MUC16 1:9#7 scFv were incubated with FLAG-tagged TRAIL (1 μg/ml) and the DISC was immunoprecipitated with anti-FLAG M2 antibody. The membrane was immunoblotted with anti-cFLIPL and with anti-cFLIPs antibodies. (E) OVCAR3 cells were transfected with control nontargeting siRNA (NT siRNA) or cFLIP siRNA pools and then incubated with TRAIL. Immunoblot analysis (24 h) was performed with anti-cFLIPL and anti-cFLIPS antibodies and showed decreased cFLIPL and cFLIPS expression in cFLIP siRNA-transfected cells. Apoptosis was qualitatively assessed by phase contrast microscopy in TRAIL treated cells (100 ng/ml; 6 h) and showed morphological changes suggestive of apoptotic cells in cFLIP siRNA-transfected cells. Representative fields from one of two independent experiments are shown. Apoptosis was also by ELISA as described in Methods and expressed as fold increased relative to control (untreated) cells with the mean of triplicates from three independent experiments ± SD. (F) Lysates from empty vector- and MUC16 CTD-expressing SKOV3 cells were immunoblotted with cFLIPL and cFLIPS antibodies.