Figure 3: IDO-Kyn metabolic circuitry mediates TRC dormancy.

(a,b) IDO1-overexpressing B16 cells were seeded in soft 3D fibrin gels for 4 days (d4) or 6 days (d6). The colony size was indicated (a) and the cell cycle was analysed (b). The relative colony size was calculated by comparing the colony size in other groups with the colony size in the Vec (d4) group, which was set to 1. Bar, 50 μm. (c) IDO1- B16 cells were transfected with IDO1 siRNA or scramble siRNA and then seeded in soft 3D 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) Kyn inhibited B16-TRC growth. B16-TRCs cultured in 3D soft gels for 2 days were treated with 150 μM Kyn for 2–4 days and at day 5, Kyn was removed from the supernatant and continued to culture for another 2 days. 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. Bar, 50 μm. (e) The same as d, but the TRCs were collected 72 h after Kyn treatment and performed cell cycle analysis. Data shown are representative of three independent experiments and error bars represent mean±s.e.m., NS, no significant difference (Student’s t-test).