Fig 2: The transcriptome of ISGs in sgScd2 Th1 cell depends on IRF3 and IRF9, but not IRF7. (A) A scatter plot of gene expression by RNA-sequencing (n = 5 per sample) compares in control and sgScd2 Th1 cells. The genes with over 1.5-fold changes are marked with black dots, and ISGs are marked with red dots (Control, n = 5; sgScd2, n = 5 biologically independent sample). (B) GSEA reveals the upregulation of the ISGs in sgScd2 Th1 cells. Genes are ranked into an ordered list on the basis of fold change in control and sgScd2 Th1 cells. Genes below the picture indicate leading edge subset. (C) Western blot analysis of SCD2 from control, sgScd2, sgScd2/sgIrf3, sgScd2/sgIrf7 and sgScd2/sgIrf9 Th1 cells. The summary of relative intensity was shown. Band intensity was determined by image j. (D) The Venn diagram showed overlaps and differences between 1.5-fold increased genes in sgScd2, sgScd2/sgIrf3, sgScd2/sgIrf7 or sgScd2/sgIrf9 Th1 cells compared to control Th1 cells. (E) A heat map depicts the gene relevant to (B). (F–H) A scatter plot of gene expression by RNA-sequencing (n = 5 per genotype) compares sgScd2/sgIrf3 (F), sgScd2/sgIrf9 (G) or sgScd2/sgIrf7 (H) Th1 cells against control Th1 cells. (I) GSEA reveals the upregulation of the ISGs in sgScd2/sgIrf7 Th1 cells. Genes are ranked into an ordered list on the basis of fold change in control and sgScd2/sgIrf7 Th1 cells. Genes below the picture indicate leading edge subset. Three experiments were performed and showed similar results (C). Data represent mean ± SD (one-way ANOVA test followed by Tukey’s post-hoc test for multiple comparisons, P****<0.0001).

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 IFNα 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: IRF9 does not regulate inflammatory signaling but does regulate IL-6 production in the colon. (A) Immunoblot analysis of phosphorylated and total IκBα (pIκBα and IκBα, respectively), phosphorylated and total p38 (p-p38 and p38, respectively), IRF9, and GAPDH (loading control) in colon lysates from wild type (WT) and Irf9−/− mice. Each lane corresponds to an individual mouse. (B) Levels of inflammatory cytokines in the colon of WT (n = 7–9) and Irf9−/− (n = 7–8) mice 14 days after AOM injection. (C) Levels of inflammatory cytokines in the serum of WT (n = 8–10) and Irf9−/− (n = 8–10) mice 14 days after AOM injection. Each symbol represents an individual mouse in (B,C). ns, not statistically significant. *** p < 0.001, **** p < 0.0001. Two-tailed t-test was used in (B,C). Data are represented as mean ± SEM in (B,C).

Fig 5: IRF9 drives STAT3 activation. (A) Representative images of pSTAT3 staining of colon tissues from AOM/DSS-treated wild type (WT) and Irf9−/− mice 14 days after AOM injection. Tissues were counterstained with hematoxylin. (B) Immunoblot analysis of phosphorylated and total STAT3 (pSTAT3 and STAT3, respectively) and IRF9 in the colon of WT and Irf9−/− mice 14 days after AOM injection. Each lane corresponds to an individual mouse. (C) Immunoblot analysis of pSTAT3 and STAT3 in the colon of ApcMin/+ and ApcMin/+Irf9−/− mice at day 70. Each lane corresponds to an individual mouse. (D) Immunoblot analysis of pSTAT3 and STAT3 in WT and Irf9−/− bone marrow-derived macrophages (BMDMs) stimulated with poly(I:C) (P (I:C)) or LPS for the indicated time. (E) Immunoblot analysis of pSTAT3 and STAT3 in WT and Irf9−/− BMDMs stimulated with IL-6 for the indicated time. Scale bar, 2 mm for whole scan and 500 μm for proximal, distal, and middle colon.