Fig. 5

Mutations in the PsK domain compromise hMLKL-driven necroptosis in cells. a MLKL expression in parental U937, edited MLKL^−/− U937, and MLKL^−/− U937 cells reconstituted with a doxycycline-inducible wild-type hMLKL construct were assessed by western blot. Loading control: anti-actin reprobe. b Sensitivity of parental U937, edited MLKL^−/− U937, and MLKL^−/− U937 reconstituted with doxycycline-inducible wild-type hMLKL to apoptotic (TS) and necroptotic (TSI) stimuli was assessed by PI uptake and flow cytometry. Data represent mean ± SEM of three independent assays. c Sensitivity of MLKL^−/− U937 cells reconstituted with doxycycline-inducible wild-type or mutant hMLKL to necroptotic death assessed at 6, 12, and 24 h post-TSI treatment by PI uptake and flow cytometry. Individual data points are plotted as circles. d Parental U937 and MLKL^−/− U937 reconstituted with wild-type hMLKL or the E351K, E258K, T357E/S358E, E258K/E351K, or D107A/E111A mutants were assessed for oligomer formation by Blue Native PAGE post-TSI treatment at the indicated time points. Separation into cytoplasmic (C) and membrane (M) fraction was validated by SDS-PAGE western blots for VDAC1 (membrane) and GAPDH (cytoplasmic). All blots are representative of ≥2 independent experiments. e Sensitivity of MLKL^−/− U937 cells reconstituted with doxycycline-inducible wild-type or mutant T357E/S358E, T357E/S358D, T357A/S358A, T357E/S358E/E351K, T357A, T357D, S358A, and S358E hMLKL to necroptotic death assessed at 6, 12, and 24 h post-TSI treatment by PI uptake and flow cytometry. Data in c and e represent mean±SEM of two independent assays of duplicate cell lines, except for T357E/S358D, which represents mean ± SD of two independent assays on a single line. Individual data points are plotted as circles. f Sensitivity of parental HT29, MLKL^−/− HT29, and MLKL^−/− HT29 cells reconstituted with doxycycline-inducible wild-type or mutant T357E/S358E, T357E/S358D hMLKL to necroptotic death at 48 h post-TSI treatment. Data represent mean±SEM of two independent assays on 4 (wild-type) or 2 (mutant) cell lines. Data for parental HT29 cells from a single experiment are shown for comparison. Individual data points are plotted as circles. g Streptavidin-binding peptide (SBP)-tagged hRIPK3 kinase domain immobilized on a streptavidin chip showed robust binding to wild-type, but not T357E/S358E, hMLKL (analyte applied over 0–1 μM) by surface plasmon resonance. Data are representative of four independent experiments