Figure 3.
Reconstitution of RIPK3, but not of RIPK3-KD, promotes spontaneous MLKL phosphorylation and necroptotic cell death. (a) Overexpression of RIPK3 and RIPK3-KD (D160N) in melanomas. Retroviruses expressing human RIPK3 or RIPK3-KD, or vector controls were used for transduction of A375, RPM-EP, and IGR melanoma cells. Transduced cells were selected and analysed for RIPK3 overexpression, as well as for MLKL, p-MLKL, and β-tubulin as loading control. Five micrograms of total protein lysates were separated with 4–12% NuPAGE gradient gels before detection of respective proteins by western blot analysis. One representative experiment of a total of three independent performed experiments is shown. (b–d) RIPK3 but not RIPK3-KD promotes caspase-independent cell death. (b) A375 cells with expression of control (vector), RIPK3, and RIPK3-KD were either non-stimulated or pre-stimulated with zVAD-fmk (10 μM) or IAP antagonist (100 nM) alone or in combination for 1 h before stimulation or costimulation with CD95L (0.5 U/ml) for 18–24 h. Surviving attached cells were quantified with crystal violet assay as described in Materials and Methods. Summary of multiple independent performed experiments (four experiments) including the S.E.M. of all experiments is depicted. (c) For phosphatidylserine (PS) externalization and cell death quantification, AnnexinV/PI double stainings was performed. A375 cells with expression of control (vector), RIPK3, and RIPK3-KD were pre-stimulated with DMSO/Ethanol (control), 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 14 h. Externalization of PS and cell death were 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. (d) A375 cells with expression of the respective constructs were stimulated as described in c followed by qualitative characterization of cell death by fluorescent microscopy after Hoechst/Sytox green double staining as detailed in the Materials and Methods section. One of two independent experiments is shown (L, through Light-microscopy; H, Hoechst staining; S, Sytox green staining)