Figure 4.

Kynurenine, the main catabolite, is indispensable for IDO1-mediated cellular senescence and chemoresistance. (A) Schematic diagram showing the process to study the effects of conditioned medium (CM) collected from IDO1-overexpressing CRC cells or IDO1-silencing CRC cells on cell senescence of CRC cells. (B, C) The CM derived from HCT-116 cells with IDO1 silencing (B) or IDO1 overexpression (C) was collected and boiled at 100°C for 1 h and removed precipitate by centrifugation. HCT-116 cells were supplemented with control CM or boiled CM for 5 days and analyzed by SA-β-gal staining. (D) ELISA was used to determine the contents of Kyn in the indicated CM. (E) MTT assay was performed to determine cell viability of HCT-116 and HCT-8 cells exposed to Kyn (100 μM) and various concentrations of 5-FU alone or in combination for 48 h. (F) HCT-116 cells were treated with Kyn (100 μM) in the presence or absence of 5-FU (20 μM) and analyzed by colony formation assay. (G) CRC cells were treated with or without Kyn (100 μM) in the presence of 5-FU (20 μM) and then analyzed by SA-β-gal staining. (H) Western blot was performed to detect protein levels of p53 and p27 in IDO1-silencing HCT-116 cells after exposure to 100 μM Kyn. (I) The IDO1-silencing HCT-116 cells were treated with Kyn (100 μM) in the presence of the indicated concentrations of 5-FU and the cell viability was measured by MTT assay. *P < 0.05; **P < 0.01.