Figure 4: TRC dormancy is regulated by Kyn-AhR-p27 pathway.

(a) Kyn promoted the translocation of AhR from the cytosol to nucleus by immunostaining assay. Bar, 10 μm. (b) Cell fraction of cytosol (C) and nucleus (N) was analysed by western blot. (c) Stably IDO1-B16 cells were transfected with scramble or AhR siRNA and seeded in 3D soft fibrin gels and cultured for the indicated days. The colony size is presented photographically and graphically. The relative colony size was calculated by comparing the colony size in groups with that in the Vec (d4) group, which was set to 1. Bar, 50 μm. (d) B16 cells were stably transfected with scramble shRNA, AhR shRNA1 or AhR shRNA2, and cultured in soft 3D fibrin gels with or without Kyn (150 μM) for 48 h. Scramble-B16 TRCs were used as control. The colony size is presented photographically and graphically after Kyn treatment for 48 h. The relative colony size was calculated by comparing the colony size in groups with that in the ShAhR/PBS group, which was set to 1. Bar, 50 μm. (e) B16 TRCs were treated with different concentration of Kyn. AhR activity was determined by luciferase assay. (f) B16-TRCs were treated with Kyn (150 μM) in the presence or absence of DMF (20 μM). The expression of p27 was determined by real-time PCR (left) and western blot (right). (g) the expression of p27 in TRCs or control B16 cells with or without IFN-γ treatment. (h) Kyn enhanced the luciferase activity of p27 promoter. B16-TRCs were transiently transfected with a luciferase reporter plasmid of pGL3/p27 promoter with or without Flag-AhR in the presence or absence of Kyn for 24 h. Data shown are representative of three independent experiments and error bars represent mean±s.e.m. (Student’s t-test).