Fig. 6. The IDO1/TDO–Kyn–AhR signaling pathway modulated glioma cell migration and invasion via AQP4.

a Western blot analysis of the effects of IDO1 and TDO on AQP4 expression in U87MG cells. n = 3 per group. b Western blot analysis of the effects of Kyn, SR-1 (AhR inhibitor), or Kyn + SR-1 on the expression of AhR and AQP4 in U87MG cells. n = 3 per group. c qPCR analysis of the effects of Kyn, SR-1, or Kyn + SR-1 on the mRNA expression of AhR, CYP1A1, CYP1B1, and AQP4 in U87MG cells. n = 3 per group. d Schematic diagram of predicted AhR-binding sites (DREs) in the AQP4 promoter. e ChIP analysis of AhR binding to the AQP4 promoter in U87MG cells treated with Kyn. ChIP assays were performed with control IgG or anti-AhR antibody. Immunoprecipitated DNA was examined using qPCR and primers specific for the AQP4 promoter. Samples were normalized to the amount of input DNA. n = 3 per group. f Western blot analysis of the expression of AQP4 in AQP4-overexpressing (OE-AQP4) or knockdown (si-AQP4) U87MG cells. n = 3 per group. g Migration and invasion assays of U87MG cells under different conditions (magnification, ×200; scale bar, 100 μm). n = 3 per group. h Morphologic changes in U87MG cells upon different treatments were recorded by phase-contrast microscopy (left). Cell area per cell was quantified (right) (magnification, ×400; scale bar, 50 μm). n = 5 per group. i Phalloidin-iFluor 488-labeled F-actin (green) in U87MG cells upon different treatments was recorded by fluorescence microscopy. DAPI (blue) was used for nuclear staining (left). Fluorescence intensity of F-actin per cell was quantified (right). (Magnification, ×400; scale bar, 50 μm) n = 5 per group. The designation of the different treatments is described in the Materials and methods section. Statistical significance was determined by one-way ANOVA followed by Dunnett’s test. Data are presented as the mean ± SEM. **p < 0.01; ***p < 0.001.