Fig 2: ERRa regulates TBK1-IRF3 complex formation.(A) Immunoprecipitation analysis in 293T cells transfected with Flag-IRF3 and HA-TBK1 or HA-IKKe in the presence or absence of Myc-ERRa. (B) In situ PLA assay of the IRF3-TBK1 complex in 293T cells infected with VSV (MOI = 1.0) in the presence or absence of 2.5 µM XCT790 for the indicated times. TBK1-IRF3 complex, red; nuclei, blue. (C) Immunoprecipitation analysis in WT and ERRaCRISPR-/- cells infected with VSV (MOI = 1.0) for the indicated times. (D) Immunoblotting analysis in WT and ERRa-KO BMDMs infected with VSV (MOI = 1.0) for the indicated times. (E) Native gel electrophoresis (Native-PAGE) or SDS–PAGE anlaysis in WT and ERRaCRISPR-/- cells transfected with the indicated expression plasmids and infected with VSV (MOI = 1.0) for the indicated times. (F) Immunoprecipitation analysis in 293T cells transfected with Flag-IRF3 and HA-IRF3 in the presence or absence of Myc-ERRa. ChIP assay in 293T cells transfected with Flag-ERRa and infected with VSV. PCR products were verified by agarose gel electrophoresis. (H) IFN-ß promoter luciferase activity assay in ERRaCRISPR-/- cells transfected with HA-TBK1 and Flag-ERRa or its mutants. (I) Plaque assay of VSV loads in supernatants of ERRaCRISPR-/- cells transfected with Flag-ERRa or its mutants and infected with VSV (MOI = 1.0) for 12 h. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.

Fig 3: Gene deletion of SCD2 resulted in the translocation of IRF3 and IRF9 from cytosolic to nuclear. (A, B) Western blot analysis of phospho-IRF3 (pIRF3), total IRF3 (A) and IRF9 (B) from control and sgScd2 Th1 cells. (C) qRT-PCR analyses of the relative expression of Irf7 from control, sgIrf7, sgScd2 and sgScd2/sgIrf7 Th1 cells. Relative expression (normalized to Hprt) with SD is shown. (D) Intracellular staining and flow cytometry analyzing of IRF7 in control, sgIrf7, sgScd2 and sgScd2/sgIrf7 Th1 cells. Mean fluorescence intensity (MFI) of IRF7 are shown. Summary data of three independent experiments of IRF7 expression are shown here. Each dot represents one experiment. Data are means ± SD. (n = 3 per each group biologically independent sample). (E) Western blot analysis of pIRF3, total IRF3 and IRF9 from control, sgScd2 and sgScd2/sgTmem173 Th1 cells. (F, G) Western blot analysis of pSTING, total STING (F) and pTBK1, TBK1 (G) from control, sgScd2, sgScd2/sgIrf3, sgScd2/sgIrf7 and sgScd2/sgIrf9 Th1 cells. (H) The amount of IFNa in the cell supernatant was measured by ELISA. Data are means ± SD. (I) Western blot analysis of IRF9 from control, sgScd2 and sgScd2/sgTmem173 Th1 cells. (J) Western blot analysis of pIRF3 and total IRF3 from control, sgScd2, sgScd2/sgIrf3 and sgScd2/sgIrf9 Th1 cells. (K) Western blot analysis of IRF9 from sgScd2 Th1 cells treated with 10µg/ml IFNAR neutralizing antibody. Isotype antibody was used as control. Band intensity was determined by image j and summary of three independent experiments was shown. Three technical replicates were performed with quantitative RT-PCR and relative expression (normalized to Hprt) with SD is shown (C). Three experiments were performed and showed similar results. Data represent mean ± SD (unpaired two-tailed student t tests or one-way ANOVA test followed by Tukey’s post-hoc test for multiple comparisons, P*<0.05, P**<0.01, P***<0.001, P****<0.0001). ns means “not significant”.

Fig 4: ERRa associates with TBK1, IKKe and IRF3.(A-B) IFN-ß promoter luciferase activity assay in 293T cells transfected with the indicated plasmids. (C) IFN-ß promoter luciferase activity assays in shCtrl or shERRa-2 cells transfected with the indicated plasmids. (D-E) GST pulldown assay in 293T cells transfected with Flag-TBK1 (D) or Flag-IRF3 (E). (F-H) Immunoprecipitation analysis in 293T cells transfected with Flag-ERRa and HA-TBK1 (F), HA-IKKe (G) or Myc-IRF3 (H). (I) Far-western analysis in 293T cells transfected with Flag-IRF3. In situ PLA assay of the ERRa-TBK1 complex in 293T cells infected with VSV (MOI = 1.0) for the indicated times. ERRa-TBK1 complex, red; nuclei, blue. Quantification of PLA signals per cell in Fig 4J presented relative to control cells treated with solvent. (L-M) Immunoprecipitation analysis in 293T cells transfected with the indicated plasmids. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.

Fig 5: EhaF is a secreted autotransporter translocating into the host cell cytosol during infection.a The domain organization of EhaF with an N-terminal signal peptide (residues: 1–31), a passenger domain (residues: 32–309), and a C-terminal beta domain (residues: 310–369). b Immunoblot for EhaF-FLAG in the bacterial supernatant and pellet of H2O- or IPTG-treated BL21/pEmpty-FLAG, BL21/p?SP-EhaF-FLAG (lacking signal peptide), and BL21/pEhaF-FLAG (full-length EhaF). c, d IFNß and IL-6 secretion by BMDMs treated with supernatants (25 µl) from H2O or IPTG-treated BL21/pEmpty, BL21/pEhaF, or BL21/p?SP-EhaF 30 min prior to treatment with 1 µg/ml LPS or wild-type EHEC or ?EhaF (MOI = 50) at 6 h post-treatment. e, f Agar plating for bacterial counts (e) and immunoblots for EhaF-FLAG, EEA1, Rab7, LAMP1, Na + /K + ATPase, and GAPDH (f) in the cytosolic and residual fractions of uninfected BMDM or BMDM infected with the indicated strains for 1.5 h at an MOI of 50 obtained by 0.005% digitonin fractionation. g Transmission electron microscopy of BMDMs infected with IPTG-treated BL21/pEmpty-FLAG or BL21/pEhaF-FLAG at an MOI of 50 for 1.5 h and stained with gold-conjugated anti-FLAG antibody (red arrows show FLAG labeling). Scale bar=500 nm. h Immunoblot for EhaF-FLAG, IRF3, and p65 in the elute from FLAG immunoprecipitation (FLAG IP) or in the lysates (Input) from BMDMs at 1.5 h following infection with IPTG-treated BL21/pEmpty-FLAG or BL21/pEhaF-FLAG. c, d Data (mean ± SEM) were from three independent experiments and each dot is a mean of each experiment’s technical replicates. Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-test. p < 0.05 indicated statistical significance. Multiplicity adjusted p values are presented. b, e, f, g, h Data from one experiment representative of three independent experiments is shown. Source data are provided as a Source Data file.