Figure 5: IFN-γ activated IDO-Kyn-AhR cascade crosstalks with STAT1 pathway via p27.

(a) the expression of IDO1 in control B16 cells, B16 TRCs or IFN-γ-treated B16 TRCs was determined by western blot. (b) the expression of AhR in control B16 cells, B16 TRCs or IFN-γ-treated B16 TRCs was determined by western blot. (c,d) B16 TRCs were treated with 100 ng ml⁻¹ IFN-γ for 3 days. The nuclear localization of AhR was determined by immuno-fluorescence staining (c) and western blot (d). Cytoplasmic β3-tubulin and nuclear TopBp1 were used as controls. (e) B16 TRCs were treated with IFN-γ (100 ng ml⁻¹) or IFN-γ+DMF (20 μM) for 3 days. The expression of p27 was determined by western blot. (f–h) B16 TRCs were treated with or without IFN-γ (100 ng ml⁻¹) for 3 days. Phosphorylated STAT1 in whole cell lysate (f) or cytoplasmic and nuclear fractions (g) was analysed by western blot. Immunostaining of p-STAT1 (green) and DAPI (blue) was represented (h). Bar, 10 μm. (i) B16 TRCs were treated with IFN-γ (100 ng ml⁻¹) for indicated time. Cytoplasmic and nuclear p27 was analysed by western blot. (j) B16 TRCs, stably transfected with scramble shRNA or IDO1 shRNAs, were treated with IFN-γ (100 ng ml⁻¹) for 72 h. Cytoplasmic and nuclear expression of p27 was analysed by western blot. (k) B16-TRCs were treated with IFN-γ (100 ng ml⁻¹) for 24 or 72 h and the cytoplasmic and nuclear fractions were collected. Anti-phospho-STAT1 immunoprecipitates from both cytoplasm and nucleus were immunoblotted with anti-p27 and anti-phospho-STAT1 antibodies. Images shown are representative of three independent experiments.