Figure 2.

IDO1 is also regulated by attachment-independent growth via JAK/STAT signaling

(A) Schematic diagram of the kynurenine pathway.

(B–D) Protein expression was analyzed after 24 h of (B) culture under either normoxic (20% O₂) or hypoxic (1% O₂) conditions, (C) treatment with rotenone (1 μM) or vehicle-only control, or (D) culture in media containing either glucose (Glc) (10 mM) or galactose (Gal) (10 mM).

(E and F) Cell lines were cultured in 2D or 3D conditions for 24 h and cell lysates analyzed for protein expression, and (F) band intensity of IDO1 relative to actin (loading control) was quantified with a Li-Cor infrared scanner (data from n = 4 independent experiments, p value calculated with paired 2-sided t test, bars are SDs).

(G) CFPAC-1 cells were cultured in 2D or 3D (attachment-independent) conditions for 24 h and treated with epacadostat (1 μM) or vehicle-only control for 16 h before media (extracellular) kynurenine was analyzed by LC-MS (n = 3 biological replicates, bars are SDs).

(H) CFPAC-1 and HPAF-II cells were cultured in either 2D or 3D conditions for 24 h and then treated for 16 h with JAKi or vehicle-only control (veh.) and/or human IFNγ (1 ng/mL). Cells were then lysed and protein expression analyzed.