Fig. 6: Mlkl ^K219R/K219R knock-in cells are protected from TNF and MCMV-driven necroptosis.

a Quantification of propidium iodide positive (PI⁺) primary bone marrow-derived macrophages (BMDMs) derived from two Mlkl^WT/WT and Mlkl^K219R/K219R mice. Cell death was measured following treatment with TNF (10 ng/ml), SM-164 (100 nM) and z-VAD-FMK (20 μM; TSZ) for 4 h in presence or absence of RIPK1 inhibitor (RIPK1i GSK′963, 100 nM) or RIPK3 inhibitor (RIPK3i GSK′843 2 μM). b Quantification of PI⁺ mouse dermal fibroblasts (MDFs) derived from two Mlkl^WT/WT and Mlkl^K219R/K219R mice following treatment as in a. In a, b, n = 3 wells/group. The results are representative of those from two independent experiments with three technical replicates each. Data are presented as mean ± SD. Statistical analysis shown was calculated by two-way ANOVA with Sidak’s multiple comparison test. Data from mice with same genotype was pooled together for the statistical analysis in a and b. c Western blot analysis of MLKL expression in MDFs from two pairs of Mlkl^WT/WT and Mlkl^K219R/K219R mice, following treatment with TSZ for 2.5 h or left untreated. d MLKL oligomerisation analysis. Blue native polyacrylamide gel electrophoresis (BN-PAGE) of the cytoplasmic (C) and membrane (M) fractions of MDFs from Mlkl^WT/WT and Mlkl^K219R/K219R mice, following treatment with TSZ for 2.5 h or left untreated. Shown on the right is the quantification of MLKL oligomers at the plasma membrane from two independent experiments. e Cell viability assay of Mlkl^WT/WT and Mlkl^K219R/K219R MDFs infected with wild-type (WT) and M45 mutant RHIM (M45mutRHIM) murine cytomegalovirus (MCMV^WT and MCMV^M45mutRHIM). f Measurement of viral growth. Mlkl^WT/WT and Mlkl^K219R/K219R MDFs were infected with the respective virus and the viral titre was determined by plaque assay 72 h post infection. In e and f, n = 4 and 3 wells/group, respectively. The results are representative of those from three independent experiments with three to four technical replicates each. Data are presented as mean ± SD. Statistical analysis shown was calculated by two-way ANOVA with Sidak’s multiple comparison test. Source data are provided as a Source data file.