Figure 4.
CD95L/IAP antagonist-induced necroptosis in RIPK3-re-expressing A375 cells is partially RIPK1 kinase independent and promotes MLKL phosphorylation. (a–c) CD95L/IAP antagonist-mediated necroptosis but not CD95L-induced apoptosis in RIPK3-expressing A375 cells is partially RIPK1. (a and b) Control, RIPK3-, and RIPK3-KD-expressing A375 cells were either pre-stimulated with Nec-1 (50 μM), zVAD-fmk (10 μM), or IAP antagonist (100 nM) alone or in respective combination for 1 h before stimulation or costimulation with CD95L (0.5 U/ml) for 18–24 h (a), for 14 h (b), followed by analysis with respective methods. (a) Crystal violet staining was performed as previously described. Summary of multiple independently performed experiments (four experiments) including the S.E.M. of all experiments is depicted. (b) Externalization of phosphatidylserine (PS) and cell death was analysed after Annexin V/PI double staining by FACS analysis as described in Materials and Methods section. One of two independent experiments is shown representatively. For analysis of MLKL phosphorylation, vector control, RIPK3-, and RIPK3-KD-expressing A375 cells were stimulated with IAP antagonist (1 h prestimulation with 100 nM) followed by CD95L (0.5 U/ml) stimulation for the indicated time (c). The respective cell lines were either separately pre-stimulated or co-stimulated with IAP antagonist (1 h pre-stimulation with 100 nM) and zVAD-fmk (1 h pre-stimulation with 10 μM), followed by CD95L treatment (0.5 U/ml) for 6 h (d). Phosphorylation of MLKL as well as expression of MLKL, RIPK3, RIPK1, cIAP1, actin, and β-tubulin was analysed by western blot analysis as previously described. One of three (c) and one of two (d) representative and independently performed experiments is shown