Fig 2: JMJD1A cooperates with STAT1 to promote the expression of IRF1.(A) The mRNA level of CCL8 was decreased in JMJD1A knock-down CT26 cells with heat-killed C. rodentium treatment. (B) The mRNA level of CCL8 was decreased in the IRF1 knock-down CT26 cells with heat-killed C. rodentium treatment. (C) The mRNA level of IRF1 was decreased in the JMJD1A knock-down CT26 cells with C. rodentium treatment. (D) The protein levels of JMJD1A and IRF1 were decreased in JMJD1A knock-down CT26 cells by western blot with heat-killed C. rodentium (MOI = 200) treatment. (E) The protein level of JMJD1A and IRF1 were decreased in colons of WT and JMJD1A-/- mice by western blot after C. rodentium infection. (F) KEGG pathway analysis showed that Jak-STAT signaling pathway was enriched. (G) The protein levels of p-STAT1 and STAT1 were increased in the colons of mice after C. rodentium infection. (H) JMJD1A could cooperate with STAT1 to enhance IRF1 promoter activity in CT26 cells. CT26 cells were co-transfected with Renilla plasmids, murine IRF1 promoter, JMJD1A expression plasmids and STAT1 expression plasmids using Lipofectamine 2000. The luciferase and Renilla values were measured at 24 h after co-transfection. n = 3. (I) The endogenous JMJD1A and STAT1 in CT26 cells could be detected by co-immunoprecipitation using STAT1 and JMJD1A antibodies, respectively. (J) STAT1 bound to the IRF1 promoter without HKCR treatment, and the binding was increased after HKCR treatment. The affinity of STAT1 on the IRF1 promoter in CT26 cells via ChIP assay using STAT1 antibodies or IgG antibodies. (K) JMJD1A bound to the IRF1 promoter without HKCR treatment, and the binding was increased after HKCR treatment. The affinity of JMJD1A on the IRF1 promoter in CT26 cells was measured via ChIP assay using JMJD1A antibodies or IgG antibodies. (L) The binding of H3K9me2 was decreased after HKCR treatment. The affinity of H3K9me2 on the IRF1 promoter in CT26 cells was measured via ChIP assay using H3K9me2 antibodies or IgG antibodies. Data are mean ± SEM. Results are representative of three independent experiments. P values were assessed with the two-tailed Student’s t-test or nonparametric tests, or one-way ANOVA test. *P < 0.05; **P < 0.01.

Fig 3: JMJD1A cooperates with IRF1 to promote CCL8 expression.(A) JMJD1A could cooperate with IRF1 to enhance CCL8 promoter activity. CT26 cells were co-transfected with Renilla plasmids, murine CCL8 promoter reporter, JMJD1A expression plasmids or JMJD1A mutant expression plasmids, and IRF1 expression plasmids using Lipofectamine 2000. The luciferase and Renilla values were measured at 24 h after co-transfection. n = 3. (B) JMJD1A antibodies could immunoprecipitate IRF1 and IRF1 antibodies could immunoprecipitate JMJD1A. HEK293 cells were co-transfected with Flag-JMJD1A expression plasmids and Myc-IRF1 expression plasmids using Lipofectamine 2000. Co-IP analysis of the interaction of JMJD1A and IRF1 with indicated tag antibodies. n = 3. (C) JMJD1A antibodies could immunoprecipitate IRF1 and IRF1 antibodies could immunoprecipitate JMJD1A in CT26 cells. CT26 cells were treated with HKCR (MOI = 200), and cell lysis was collected. Co-IP analysis of the interaction of JMJD1A and IRF1 with indicated antibodies. n = 3. (D) JMJD1A and IRF1 bound to the CCL8 promoter without HKCR treatment, and the binding was enhanced after HKCR treatment, but the level of H3K9me2 was reduced. CT26 cells were treated with or without HKCR (MOI = 200), and ChIP assays were performed. The affinity of IRF1 on the CCL8 promoter in CT26 cells was measured by ChIP assay using IRF1 antibodies or IgG antibodies. The affinity of JMJD1A on the CCL8 promoter in CT26 cells was measured by using JMJD1A antibodies or IgG antibodies. The affinity of H3K9Me2 on the CCL8 promoter in CT26 cells was measured by using H3K9me2 antibodies or IgG antibodies. (E-H) HKCR treatment caused an increase in the chemoattraction of Raw264.7 and CTLL-2 cells, while knockdown of JMJD1A or IRF1 in CT26 cells decreased the chemoattraction of Raw264.7 and CTLL-2 cells. Graph showing number migrating toward in a chemotaxis assay. The data were normalized by the cell number of negative control (siNC) group. n = 4. (I) Schematic model for the mechanism by which JMJD1A protect mice from C.rodentium infection by upregulating CCL8 to recruit macrophages and CD4+ T cells. The image was created by “ScienceSlides 2005” plugin and Microsoft PowerPoint software. Data shown in graphs are Mean ± SEM or SD. The data were pooled from three independent experiments. P values were assessed with the two-tailed Student’s t-test or nonparametric tests, or one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.