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. 2020 Dec 3;28(5):1610–1626. doi: 10.1038/s41418-020-00690-y

Fig. 2. The protective effect of primidone against regulated cell death is RIPK1-mediated.

Fig. 2

A MDFs carrying the inducible active Mlkl mutant S345D (MlklS345D) were induced for 7 h at 37 °C with 0.5 μg/ml doxycycline (doxy) in the presence of 1 mM phenytoin or 1 mM primidone, respectively, 30 min before the addition of doxycycline. Doxycycline-induced cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. FACS dot plots of one representative experiment are shown; the adjacent graph presents the mean ± SD of three independent experiments. B Non-edited (parental) L929 cells were treated for 24 h at 37 °C with DMSO (vehicle), 10 ng/ml TNFα, 1 µM 5Z-7 or a combination of TNFα + 5Z-7 in the absence or presence of 50 µM Nec-1s and 1 mM primidone. The combination of TNFα + 5Z-7 induced a RIPK1-dependent apoptosis (RDA), which is driven by caspase-8 inhibition (+25 µM zVAD) to RIPK1-dependent necroptotic cell death. Both cell death modalities, i.e., RDA as well as RIPK1-dependent necroptosis, are blocked by primidone. One representative approach of three independent experiments is shown. C Identical approaches using CRISPR/Cas9-edited Ripk1-deficient L929 cells confirm RDA as a promoted cell death pathway in response to TNFα + 5Z-7 stimulation. Cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. Depicted is one of three independent experiments. Fig. S1C illustrates a histogram showing the mean ± SD of the three independent experiments shown in B, C. D Human Jurkat cells were stimulated for 4 h at 37 °C with 100 ng/ml Fas ligand (FasL) in the absence (vehicle) or presence of 1 mM primidone. RIPK1-independent apoptotic cell death in this setting was confirmed by the addition of the pan-caspase inhibitor zVAD. The graph shows the mean ± SD of three independent experiments. E Human HT-1080 cells were stimulated at 37 °C for 24 h with 100 ng/ml TNFα + 1 μg/ml cycloheximide (CHX) in the absence or presence of 50 µm Nec-1s and 1 mM primidone, respectively. The combination of TNFα and CHX promoted TRADD-mediated RIPK1-independent apoptosis, which was not inhibited by the addition of the RIPK1-specific inhibitor Nec-1s or by primidone. Depicted is one of three independent experiments.