Figure 6: Switching IFN-γ-mediated IDO-Kyn-AhR cascade to STAT1 signalling targets TRC dormancy.

(a,b) B16 cells (1.25 × 10³) were seeded in soft 3D fibrin gels. Two days later, IFN-γ was added for further 2 days (d2) or 4 days (d4) culture in the presence or absence of different concentration of DMF. The colony size was presented (a) and colony number was counted (b). The relative colony size was calculated by comparing the colony size in other groups with the colony size in the PBS (d2) group, which was set to 1. The data represent mean±s.e.m. (n=3). Bar, 50 μm. (c,d) The above B16 TRCs were cultured in the presence or absence of different concentration of 1-MT. The colony size was presented (c) and colony number was counted (d). The relative colony size was calculated by comparing the colony size in other groups with the colony size in the PBS (d2) group, which was set to 1. The data represent mean±s.e.m. (n=3). Bar, 50 μm. (e) control B16 cells or B16 TRCs were treated with 100 ng ml⁻¹ IFN-γ for 24 or 72 h (n=3). Caspase 3 (Cas 3), caspase 7 (Cas 7) and their cleaved forms (Cas 3F and Cas 7F) were determined by western blot. (f) B16-TRCs were treated with IFN-γ (100 ng ml⁻¹) plus 1-MT (0.2 mM) or DMF (20 μM) for 72 h (n=3). Caspases were analysed by western blot. (g) B16-TRCs were treated with IFN-γ (100 ng ml⁻¹), IFN-γ/1-MT (0.2 mM) or IFN-γ/DMF (20 μM) for 72 h. Immunostaining of p-STAT1 (green) and DAPI (blue) was shown as the representative of three independent experiments. Bar, 10 μm.