Figure 1. Reduced indoleamine 2,3-dioxygenase enzyme activity and expression under hypoxic conditions.

(A–C) Determination of the kynurenine production in different cells after IDO induction by IFN-γ in vitro. Glioblastoma cells 86HG39 (A), HeLa cells (B) or human foreskin fibroblasts (HFF) (C) were stimulated with IFN-γ (500 U/mL) or left unstimulated in IMDM cell culture medium containing additional L-tryptophan (100 µg/mL). The 72 h incubation period was carried out at normoxia (20% O₂), (10% O₂) or hypoxia (1% O₂). The kynurenine content in the cell culture supernatants was determined by optical density at 492 nm +/− SEM, using Ehrlichs reagent. A significant inhibition of kynurenine production as compared to the stimulated normoxia positive control is marked with asterisks, n = 3. (D-F) Western Blot analysis of 86HG39 (D), HeLa (E) or HFF lysates (F) stimulated with IFN-γ (0–500 U/mL) under normoxia or hypoxia for 24 h. Protein expression of IDO, phosphorylated STAT1 (pSTAT1) and β-actin was detected. (G-L) Densitometric analysis of the Western Blots shown in D–F. Relative protein expression of IDO and pSTAT1 with reference to the β-actin protein expression.