Extended Data Fig. 6: OAA prompts ETS2 activation in a MDH1-dependent manner.

a, Total, cytosolic, and nuclear fractions of THP-1 cells pre-treated with DEOAA (1 mM) for 12 h were immunoblotted with antibodies directed against ETS2, a cytosol marker (α-Tubulin), or a nucleus marker (PCNA). b, A549 cells transfected with Scr or MDH1-specific siRNAs were challenged with H1N1 (PR8) virus (MOI = 1) in the absence or presence of DEOAA (1 mM). The transcription levels of IFNB1 and ISGs were analyzed by qPCR. c, Intracellular NP vRNA levels in the sample as (b). d, Lysates of 293 T cells transfected with plasmids of Flag-MDH1, Flag-MDH2 and HA-ETS2 were immunoprecipitated with α-Flag M2 beads and immunoblotted with anti-HA. e, Luciferase activity of WT and MDH1 KO 293 T cells transfected with plasmid of TBK1-Luc in the absence or presence of DEOAA (1 mM). f, WT Flag-MDH1 or its indicated mutants purified from 293 T cells were incubated with biotin-labelled OAA in vitro, and subjected to pull-down assay using streptavidin-coated magnetic beads for further immunoblot analysis. g, Relative NADH/NAD⁺ levels of MDH1-deficent THP-1 cells reconstituted with plasmids of Flag-MDH1 and its indicated mutant. h, Extracts of THP-1 cells cultured in increasing doses of DEOAA were treated with 2 mM disuccinimidyl suberate (DSS) cross-linker and analyzed by immunoblotting. i, Extracts of 293 T cells expressing Flag-tagged WT MDH1 or its indicated mutants in the absence or presence of DEOAA (1 mM) were treated with 2 mM DSS cross-linker and analyzed by immunoblotting. j, Extracts of 293 T cells expressing Flag-tagged WT MDH1 or its indicated mutants in the absence or presence of DEOAA (1 mM) were treated with 2 mM DSS cross-linker and analyzed by immunoblotting. k, MDH1 KO 293 T cells were transfected with plasmid of TBK1-Luc, along with the vectors of encoding WT, or indicated mutant form Flag-MDH1. The cells were treated with DEOAA (1 mM) and the protein extracts were harvested for luciferase assay. l, MDH1 KO THP-1 cells expressing WT or indicated point mutated form of MDH1 were challenged with H1N1 (PR8) virus (MOI = 1) for 24 h and the samples were harvested and analyzed by ELISA. m, MDH1 KO THP-1 cells expressing WT or indicated point mutated form of MDH1 were infected with H1N1 (PR8) virus (MOI = 1) for 24 h, and the supernatants were collected for plaque assay. b,c,e,g,k–m, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-way ANOVA with Tukey’s multiple-comparison test (b) or two-tailed unpaired Student’s t test (c, e, g and k–m). a,d,f,h–j, Similar results were obtained in 3 independent biological experiments.

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