Fig. 1: METTL3 globally inhibits innate immune signaling cascade.

a qPCR analysis of Ifnb1 expression following a 12 h treatment with different triggers as indicated before overexpression of Mettl3 in RAW264.7 cells (Left). qPCR analysis of IFNB1 expression following a 12 h treatment with HBV before knockdown of METTL3 in AC12 cells (Right). n = 3 biologically independent experiments. b IFN-β promoter activity in HEK293T cells transfected with METTL3 vector upon different treatments for 12 h (Left). IFN-β promoter activity in AC12 cells transfected with siMETTL3 upon HBV treatment for 12 h (Right). n = 3 biologically independent experiments. c Western blot analysis of the proteins in innate immune signaling isolated from peritoneal macrophage of WT or Mettl3-deficient mice. The peritoneal macrophage was treated with PBS or VSV infection for 12 h before lysis. d Western blot analysis of the proteins from A549 cell line before treated with siScramble or siMETTL3 for 2 days. e–h Western blot analysis of the proteins from RAW264.7 (e), HeLa (f), Huh7 (g), and LO2 (h) cell lines before overexpression of METTL3 for 2 days. i Immunofluorescence analysis of IRF3 translocation and activation in HEK293T cells after VSV infection for 6 h or PBS treatment. The arrowed cells indicated the METTL3 overexpressed cells which inhibited the nuclear translocation and activation of IRF3. The “*” labeled cells indicated control cells. α-Tubulin indicated the cytosolic part. n = 2 biologically independent experiments. j GO and pathway enrichment analysis of upregulated genes upon Mettl3 knockout in RAW264.7 cells after VSV infection (two-tailed hypergeometric test with p < 0.05). k Two-tailed Gene Set Enrichment Analysis of the Ifnb1 signaling transcriptional signature in Mettl3 knockout relative to control groups. NES, normalized enrichment score. l Scatter plot showing the alteration of genes expression by comparing Mettl3 knockout and WT RAW264.7 cells after VSV infection for 12 h (yellow, upregulated; blue, downregulated; gray, no significant change; red triangle, ISGs). Data are representative of 2–3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by two-tailed unpaired Student’s t test (Fig. 1a, b, i). Error bars represent mean ± SEM.