GA induces gene expression of <i>Cyp1a1</i> and formation of Ahr/Arnt complex.

The CD4+CD62L+ T cells were isolated from the spleen and cultured under Th17-polarizing conditions, and peritoneal macrophages were cultured in presence of LPS. The expression of Cyp1a1 mRNA was assessed by quantitative real‐time PCR and normalized to Gapdh mRNA, and protein level was assessed by immunoblot using Actin as a loading control. (A) Relative expression of Cyp1a1 mRNA at 12 h in CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages in presence of GA (40–120 μmol/L) compared to vehicle (DMSO). (B) Immunoblot and relative protein level of Cyp1a1 at 48 h in CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages in presence of GA (40–120 μmol/L) compared to DMSO. (C) Relative expression of Cyp1a1 mRNA at 12 h in CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages electroporated with Ahr siRNA (siAhr) or non-specific siRNA (siNS) in presence of GA (80 μmol/L) compared to DMSO. (D) Immunoblot and relative protein level of Cyp1a1 at 48 h in CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages electroporated with siAhr or siNS in presence of GA (80 μmol/L) compared to DMSO. (E) Efficiency of siAhr in CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages was confirmed by immunoblot. (F) Detection of Arnt protein by immunoblot in the eluates pulled down by Ahr antibodies using CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages. (G and H) Relative expression of (G) Cyp1a1 mRNA and (H) protein in presence of DIM (25 μmol/L) or GA (80 μmol/L) in CD4+CD62L+ T cells cultured under Th17-polarizing conditions and macrophages compared to DMSO. Data were pooled from three independent experiments with three mice per experiment and shown as mean ± SD. *p < 0.05, (one‐way ANOVA); horizontal bars denote statistical comparison.