Gl2 sirna

siRNA targeting IDO1 and luciferase gene glabra 2 (GL2; GL2-siRNA) were designed and synthesized by GE Healthcare Dharmacon, Inc. The GL2-siRNA, which was not expressed in treated cells (scrambled siRNA), was used as a negative control. The sequences of the siRNA were as follows: IDO1 siRNA, 5′-GGGCUUCUUCCUCGUCUCUTT-3′ and GL2 siRNA, 5′-CGUACGCGGAAUACUUCGA-3′. These siRNAs were transfected into LLC cells with Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, LLC cells (1×10⁵/well) were seeded into 12-well plates until they reached 50–70% confluence. Before transfection, medium was replaced with 300 µl OptiMEM^® serum-reduced medium (Gibco; Thermo Fisher Scientific, Inc.). Subsequently, 1 µg IDO1-siRNA or GL2-siRNA was incubated with 2 µl Lipofectamine^® 2000 reagent in 200 µl OptiMEM^® serum-reduced medium at room temperature for 20 min, followed by addition of the mixture to the cells that were gently agitated to distribute the mixture uniformly for 24 h.