%0 Journal Article
%T Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis
%A Alexander Poltorak
%A Amy Y. Tang
%A Anton Buzdin
%A Beiyun C. Liu
%A Chi G. Weindel
%A Hayley I. Muendlein
%A Irina Smirnova
%A Joseph Sarhan
%A Katherine A. Fitzgerald
%A Maxim Sorokin
%A Vladimir Ilyukha
%J Archive of "Cell Death and Differentiation".
%D 2019
%R 10.1038/s41418-018-0122-7
%X 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 IFN¦Â/ml). d Treatment scheme for overnight or 1£¿h treatment of BMDMs with 2.5£¿IU/ml of recombinant IFN¦Â. 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 IFN¦Â 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£¿¦ÌM) 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£¿¦ÌM/ml). In all cases of IFN¦Â pre-treatment (overnight or 1£¿h), the IFN¦Â is washed away before addition of experimental conditions. Time point cytotoxicity quantification£¿¡À£¿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
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329789/