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. 2009 Dec 28;187(7):1037–1054. doi: 10.1083/jcb.200904158

Figure 2.

Figure 2.

cIAPs specifically block CD95L-induced cell death of MEFs and human SCCs. (A) Transformed single knockout or DKO MEFs and respective control wild-type (WT) MEFs were treated with 10 ng/ml CD95L or 500 ng/ml TRAIL for 24 h in the presence or absence of 500 nM of the IAP antagonist (left). Cells were stained with PI and analyzed by flow cytometry. Four independent cIAP1 and -2 DKO MEFs were infected with inducible mouse cIAP1 or Flag-cIAP2 and induced with 10 nM 4-HT for 24 h. Cells were then treated with CD95L and TRAIL for 24 h in the presence or absence of the IAP antagonist, stained with PI, and analyzed by flow cytometry. The mean + SEM is shown throughout. (B) DKO 1.5 MEFs, infected with inducible mouse cIAP1 and Flag-cIAP2, were induced for 24 h with 4-HT. cIAP1 or Flag-cIAP2 expression was analyzed by Western blotting. β-Actin served as a loading control. (C) DKO 2.7 and DKO 3.1 MEFs were infected with inducible mouse XIAP and induced with the indicated concentrations of 4-HT for 24 h. Induction of XIAP was verified by Western blot analysis. Cells were treated with 20 ng/ml CD95L for 24 h in the presence or absence of 500 nM of the IAP antagonist, stained with PI, and analyzed by FACS. The mean + SEM of a total of six independent experiments is shown. (D) HaCaT cells were transduced with control vector or shRNA against cIAP1 or -2 or both. Western blot analysis shows expression of cIAP1 and -2. β-Actin served as a loading control. Subsequently, cells were analyzed for sensitivity to 2.5 U/ml CD95L in the presence or absence of 100 nM of the IAP antagonist 24 h later by crystal violet assay. (E) HaCaT cells were transduced with lentiviral control vector or inducible cIAP2 as previously described (Diessenbacher et al., 2008). Cells were induced with 4-HT as indicated and then treated with 2.5 U/ml CD95L for 24 h in the presence or absence of 100 nM of the IAP antagonist. Viability was examined by crystal violet assay 24 h later. (D and E) Mean + SEM of three independent experiments is shown. MM, molecular mass.