Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis

Constitutive type I interferon (IFN-I) signaling is required for initiation of necroptosis. a, b Propidium iodide incorporation as a readout of cytotoxicity, measured every 15？min following LPS/zVAD treatment of BMDMs of indicated genotype. c Western blot of indicated proteins of resting BMDMs from B6 and Ifnb？/？ with or without overnight interferon treatment (2.5？IU IFNβ/ml). d Treatment scheme for overnight or 1？h treatment of BMDMs with 2.5？IU/ml of recombinant IFNβ. e Viability of B6 and Ifnb？/？ BMDMs measured 6？h after LPS /zVAD treatment. f Treatment scheme for overnight or 1？h antibody treatment of B6 BMDMs. g Western blot for indicated proteins from B6 BMDMs treated with antibodies as in (f) before stimulation with 200？IU/ml IFNβ for 30？min. h, i Propidium iodide incorporation of B6 BMDMs treated with LZ and antibody pre-treatment for 36？h (h) or 1？h (i) as in (f). j Propidium iodide incorporation over 22？h of B6 BMDMs treated with LPS/zVAD or TNF (50？ng/ml)/zVAD (50？μM) and Ifnb？/？ BMDMs treated with TNF/zVAD. k, l Cytotoxicity measurement at 7？h (k) or 20？h (l) from B6 and Ifnb？/？ BMDMs treated with TNF/zVAD with IFN pre-treatment as in (d). All LPS/zVAD treatments were: LPS (10？ng/ml) and zVAD (50？μM/ml). In all cases of IFNβ pre-treatment (overnight or 1？h), the IFNβ is washed away before addition of experimental conditions. Time point cytotoxicity quantification？±？SD from three independent experiments compared using student two tailed t-test: ns non-significant (p？>？0.05); *p？<？0.05; **p？<？0.01; ***p？<？0.001. All western blot and kinetic cytotoxicity data are representative of three or more experiments. See related supplementary Figure