Fig 2: Interferon receptor signaling is required for MLKL phosphorylation in B6 BMDMs a western blots of pIRF3 (S396), total IRF3, RIP1, and GAPDH from unstimulated, 30 min LPS or LPS/zVAD stimulated BMDMs from B6, Ifnb−/−, Ticam1−/− and Myd88−/− mice. b Western blots of indicated proteins from B6 and Ifnb−/− BMDMs with or without overnight 2.5 IU/ml IFNβ treated with LPS/zVAD. c Western blots of RIP1 and RIP3 kinases in BMDMs of indicated genotypes in NP40 soluble or insoluble fractions. d Western blots of indicated proteins from B6, Ifnb−/−, and Sting−/− BMDMs treated with LPS/zVAD for indicated duration of time. e–f Western blots of phospho MLKL (pMLKL), MLKL and GAPDH from B6 and Infb−/− BMDMs that were untreated or primed with 2.5 IU IFNβ/ml overnight. (e) or 1 h (f) and treated with LPS/zVAD. g Non-reducing western blot featuring MLKL oligomers from B6 and Sting−/− BMDMs (with or without 2.5 IU/ml IFNβ overnight) treated with LPS /zVAD for indicated times. h B6 and MOLF BMDMs were treated with LPS, LPS/zVAD or LPS/zVAD/Nec-1 for indicated durations and analyzed for indicated proteins by western blotting. Nec-1 was used at a concentration of 30 μM. i Non-reducing western blot featuring MLKL oligomers from B6 and MOLF BMDMs treated with LPS /zVAD for indicated times. 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. All western blot data are representative of three or more independent experiments. See related supplementary Figure 4

Fig 3: Cytosolic DNA sensing via cGAS and STING is required for constitutive interferon production. a Quantitative PCR of Isg15 and western blot of indicated proteins (b) from resting B6, Ifnb−/−, Ifnar−/−, Sting−/−, and cGAS−/− BMDMs that were untreated or pre-treated with IFNβ overnight (2.5 IU IFNβ/ml). c Propidium iodide incorporation 6 h following LPS /zVAD treatment of BMDMs, either not treated with IFN (unprimed), pre-treated with 2.5 IU IFNβ/ml overnight or 1 h before LPS treatment. d Twenty-two hours time course of propidium iodide incorporation of B6, Sting−/−, Ifnb−/−, BMDMs stimulated with LPS/zVAD. e Western blots of pSTAT1 and STAT1 from BMDMs of indicated genotype either unstimulated or 2 h LPS/zVAD stimulated. f B6 and Sting−/− BMDMs pre-treated 1 h with MAR1 IFNAR blocking or isotype control antibody and monitored for LPS/zVAD-induced cytotoxicity. g Quantitative PCR of ISG mRNA relative to GAPDH mRNA from resting B6, Ifnar−/− and MOLF BMDMs. h Propidium iodide incorporation 6 h following LPS /zVAD treatment of B6, Ifnar−/− and MOLF BMDMs with or without overnight interferon pre-treatment (2.5 IU IFNβ/ml). i Propidium iodide incorporation during 12 h of stimulation with LZ of B6 and MOLF BMDMs with or without 1 h MAR1 IFNAR blocking antibody pre-treatment. j Quantitative PCR of MX1 mRNA relative to GAPDH mRNA from resting BMDMs of indicated genotype without overnight 2.5 IU IFNβ/ml. k Propidium iodide measurement over 12 h of LPS/zVAD treatment of B6, N10 congenic B6 StingMOLF/MOLF, and MOLF BMDMs. 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 is 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 3

Fig 4: Critical threshold of MLKL expression determines oligomerization potential. a RNA sequencing of resting BMDMs showing genes downregulated in Ifnar-/-, Sting-/-, StingMOLF/MOLF, and MOLF compared to B6 (b) and significantly higher in expression compared to B6. c Western blot of ZBP1 in non-treated or O/N 2.5 IU/ml IFNß treated B6 and Ifnb-/- BMDMs. d Six hours time course of viability of LPS/zVAD treated B6, Zbp1-/-, and Ifnar-/- BMDMs. e Western blot of MLKL in non-treated or O/N 2.5 IU/ml IFNß treated B6 and Mlkl+/- BMDMs. f Nine hours time course of viability of LPS/zVAD treated B6 and MLKL+/- BMDMs. g, h Non-reducing western blot featuring MLKL oligomers from BMDMs of indicated genotype treated with LPS /zVAD either non-IFNß primed (g) or treated overnight with 2.5 IU/ml IFNß (h). i High-content imaging of endogenous MLKL in PFA fixed B6 and Ifnb-/- BMDMs treated 2 h with LPS/zVAD. j, k B6 and Ifnar-/- BMDMs transduced with either empty virus or MLKL over expressing virus and analyzed for LZ-induced cytotoxicity (j) and non-reducing western blots for MLKL oligomerization and phosphorylation (k). All LPS/zVAD treatments were: LPS (10 ng/ml) and zVAD (50 µM/ml). In all cases of IFNß pre-treatment (overnight or 1 h), the IFNß is washed away before addition of experimental conditions. MLKL signal compared using student two tailed t-test: ns is 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 5

Fig 5: MDA5 and MAVS are required for IFN-induced IFN-ß production, but not cell lethality, after ADAR deletion. a, b Cell viability of control and MDA5-deficient (a) or MAVS-deficient (b) HCC366 cells was assessed by crystal violet staining 8–13 days after GFP or ADAR KO with CRISPR-Cas9. A representative image of crystal violet staining (top) and quantitation of cell viability (bottom) from two independent biological replicates (for both a and b) are shown. Cell viability values were normalized to the GFP sg2 control #2 within each group of isogenic cell lines. c IFN-ß secretion by control or ADAR1-deficient A549 cells was measured by ELISA after treatment with either vehicle or IFN-ß (10 ng/mL) for 24 h. NCI-H1437 cells harbor a homozygous deletion of the IFNB1 locus. Technical replicates from one representative experiment are shown. Three independent biological replicates were performed for A549 cells and one experiment was performed for NCI-H1437 cells. d Immunoblots showing MDA5 and MAVS protein levels in control (GFP sgRNAs) and ADAR1-deficient A549 cells 24 h after treatment with vehicle or IFN-ß (10 ng/mL). ß-Actin served as a loading control. One representative immunoblot from two independent biological replicates is shown. e IFN-ß secretion by the indicated A549 cells was measured by ELISA after treatment with vehicle or IFN-ß (10 ng/mL). Technical replicates from one representative experiment out of two independent biological replicates are shown. f Cell viability of the indicated A549 cells from e was assessed by cell counting 2 days after treatment with vehicle or IFN-ß (10 ng/mL). Cell viability values were normalized to the GFP sg2 control #2 within each group of vehicle or IFN-ß-treated isogenic cell lines. Two independent biological replicates are shown. Error bars represent standard deviation in all graphs