Figure 8.
TWEAK sensitizes to CD95L-induced cell death in a RIP1-dependent manner and is negatively regulated by cFLIPL but not cFLIPS. (A) HaCaT and MET1 cells were either prestimulated with 0.5 ng/ml TWEAK for 2 h or 100 nM of the IAP antagonist (Ant) for 30 min and then treated with 2.5 U/ml CD95L. In the same experiments, HaCaT cells were treated with 10 µM zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and 0.5 ng/ml TWEAK for 2 h. Subsequently, cells were stimulated with 2.5 U/ml CD95L. Viability was analyzed by crystal violet assay after 18–24 h. (B) TWEAK leads to rapid down-regulation of cIAP1 and -2 expression in HaCaT. Cells were stimulated with 0.5 ng/ml TWEAK for the indicated time. Subsequently, total cellular lysates were analyzed for expression of cIAP1 or -2 by Western blotting. β-Tubulin served as a loading control. MM, molecular mass. (C) Stable knockdown of RIP1 protects HaCaT cells from CD95L-induced cell death in the presence of TWEAK. Transduced HaCaT cells as shown in Fig. 3 C were prestimulated for 2 h with 0.5 ng/ml TWEAK or diluent alone, subsequently stimulated with CD95L for 18–24 h, and assayed by crystal violet assay. The mean ± SEM of three independent experiments is shown. (D) cFLIPL but not cFLIPS protects HaCaT cells from CD95L-induced cell death in the presence of TWEAK. cFLIPL, cFLIPS, and vector-transduced control HaCaT as specified in Fig. 6 C were prestimulated with 10 µM zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and 0.5 ng/ml TWEAK for 2 h or diluent alone. Cells were then stimulated with 2.5 U/ml CD95L, and viability of cells was analyzed by crystal violet assay after 18–24 h. (A and D) The mean + SEM of three independent experiments is shown.