Fig. 2 |. STAT1, STAT3 and miR-29 control AHR expression in TAMs.

a, qPCR analysis of cytokine genes in GL261 cells (n = 3 technical replicates). b, Production of IFN-β (n = 4 independent samples) and IL-6 (n = 3 independent samples) in control media (RPMI) and TCM from GL261 cells as assessed by ELISA. Unpaired two-tailed t-test. c,d, Ahr expression in BMDMs treated with anti-IFN-β antibody or STAT1 or STAT3 inhibitor for 15 min and stimulated with RPMI or TCM from GL261 cells for 24 h (n = 3 technical replicates). e, STAT1 and STAT3 binding sites in the Ahr promoter. The arrows indicate primers designed to study STAT1 (sites 1 and 2) and STAT3 (site 3) recruitment. f, ChiP analysis of STAT1 recruitment to the Ahr promoter in BMDMs stimulated with TCM (n = 3 technical replicates). g, Luciferase activity in HEK293 cells transfected with a luciferase reporter construct driven by the Ahr promoter (pAhR-Luc) and a construct encoding constitutively activated STAT1 (n = 3 technical replicates). h, STAT3 recruitment to the Ahr promoter in BMDMs stimulated with TCM as assessed by ChIP (n = 3 technical replicates). i, Luciferase activity in HEK293 cells transfected with a luciferase reporter driven by the AHR promoter (pAhR-Luc) and a construct encoding constitutively activated STAT3 (n = 4 technical replicates). j, Predicted miR-29 binding sites in the 3′ UTR of AHR. The asterisks indicate nucleotides mutated for the luciferase reporter assay. k, miR-29a and miR-29b expression determined by qPCR in TAMs (Lin^Neg CD45⁺ CD11b⁺) sorted from the brain of naive (Control) or GL261 cell-implanted (GBM) WT mice (n = 3 biologically independent samples). l, Correlation between patient survival and miR-29a, miR-29b and miR-29c expression levels (high or low) in GBM based on TCGA data (n = 91 biologically independent samples). Log-rank test was used for statistical analysis. m,n, miR-29b ectopic expression suppresses Ahr mRNA (m) and AHR protein (n) expression in RAW264.7 macrophages (n = 3 biologically independent samples). The immunoblot images are cropped; full scans are shown in Supplementary Fig. 2a. o, Luciferase activity measured 48 h post-transfection of an AHR 3′-UTR-driven luciferase reporter co-transfected with a miR-29b mimic (miR-29b) or control oligonucleotides (control) (n = 3 biologically independent samples). The following luciferase reporters were used: empty plasmid (Empty), plasmid with intact human AHR 3′-UTR (hAHR) and plasmid with human AHR 3′-UTR containing miR-29b mutant binding site (hAHRmut). Data are representative of two (c, d, f, h, i, k, m–o) or three (a) independent experiments, with similar results obtained. Data in a–d, f–i and k–o are shown as mean ± s.e.m. P values were determined by two-sided Student’s t-testa (b, g, i, k and n) or one-way ANOVA (c, d, f, h, m and o).