c-FLIP is required for macrophage survival. (A) Expression of c-FLIP_L and c-FLIP_S isoforms in wild-type BMDM as determined by Western blot. (B,C) Macrophage death after in vitro deletion of c-FLIP. (B) BMDM from c-FLIP^f/f ER-Cre mice were cultured with ethanol EtOH or 4-hydroxytamoxifen (4-OHT) for 4 days. The frequency of dead cells after deletion of c-FLIP was quantified by staining with trypan blue. Cells were imaged in complete RPMI using a Zeiss Axovert 200 (20×/0.30 NA objective lens). Images were obtained using an AxioCam MRC camera and AxioVision Rel. 4.8 software. Black bars represent c-FLIP^f/f ER-Cre cells treated with EtOH and white bars represent c-FLIP^f/f ER-Cre cells treated with 4-OHT. (Triplicate values from a single experiment; **P < .01) (C) BMDM from c-FLIP^f/f or c-FLIP^f/f ER-Cre mice were cultured with EtOH or 4-OHT for 4 days. The number of live cells remaining after deletion of c-FLIP was quantified by trypan blue exclusion. Black bars represent c-FLIP^f/f cells and white bars represent c-FLIP^f/f ER-Cre cells. Triplicate values from a single experiment; ***P < .001. (D) Relative expression levels of c-FLIP_S and c-FLIP_L mRNA in surviving c-FLIP^f/f ER-Cre BMDM after treatment with 4-OHT. Expression levels were quantified by real-time PCR using Cyclophilin A as an internal control. Black bars represent c-FLIP^f/f ER-Cre cells treated with EtOH and white bars represent c-FLIP^f/f ER-Cre cells treated with 4-OHT. Triplicate values from a single experiment (**P < .01; ***P < .001; error bars represent standard deviations; NS, not significant).