Figure 4.
RIP1 is an important regulator of DL-mediated cell death in the absence of cIAPs. (A) RIP1 knockout (KO) or wild type (WT) MEFs were stimulated with 10 ng/ml CD95L or 10 ng/ml CD95L and 500 nM of the IAP antagonist for 24 h and then assayed for cell viability using PI and flow cytometry. The mean + SEM of a minimum of three independent experiments is shown. (B) The combination of caspase inhibitor zVAD-fmk and RIP1K inhibitor Necrostatin-1 completely protects HaCaT cells from DL-mediated cell death in the presence of the IAP antagonist. HaCaT cells were separately prestimulated with 10 µM zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and 100 nM of the IAP antagonist for 30 min, followed by stimulation with 50 ng/ml TRAIL or 2.5 U/ml CD95L for 18–24 h and subsequent crystal violet assay. Mean + SEM of three (TRAIL) or six (CD95L) independent experiments is shown. (C) Transformed cIAP1 and -2 DKO 2.3 and 2.7 MEFs were treated with 10 ng/ml CD95L for 24 h in the presence or absence of 10 µM QVD and/or 50 µM Necrostatin-1 (Necro). Respective control wild-type MEFs were treated as the DKO MEFs but in the presence or absence of the IAP antagonist (Ant). Subsequently, cells were harvested, and an aliquot was replated in 6-well plates and cultured for another 4 d followed by crystal violet staining of colonies.