Fig. 2

Regulation of IL-1β-mediated IDO1 induction in 3D spheroid model of lung adenocarcinoma cells. A RT-qPCR measurement of IDO1 mRNA in H1792 and HCC827 monolayer cells and spheroids generated in 1% low attachment dishes, in control or IL-1β containing medium for 5 days. mRNA levels were normalized to 18s mRNA and represented as fold change over control treatment. B Western blot for IDO1 protein in H1792 and HCC827 monolayer and spheroid cultures ± 5 ng/mL IL-1β for 5 days. GAPDH was used as the loading control. C Viability of H1792 and HCC827 spheroids stimulated with 5 ng/mL IL-1β ± 20 nM Epacadostat (IDOi) for 5 days was measured with CellTiterGlo 3D assay (n = 4 spheroids). Luminescence was measured and viability is represented as relative luminescence units (RLU). D IL-1β induces IDO1 expression within the cells of lung adenocarcinoma spheroids. HCC827 spheroids were grown in low attachment conditions in vehicle control or 5 ng/ml IL-1β for 5 days were immunostained for IDO1 (Texas red, red) and nucleus (DAPI, blue). E IL-1β increases the percentage of IDO1⁺ cells in the spheroid. The number of IDO1 + cells were counted and represented as a percentage of the total cell population for n = 4 images. Each image of spheroid counted contained between 60—185 total cells. Error bars represent ± SD of 3 biological replicates; *P ≤ 0.05, ** ≤ 0.005, *** ≤ 0.0005