Fig. 4

a Western blot analysis of IDO and HO-1 proteins following the knockdown of IDO expression by its specific siRNA before and after the exposure of CLS-354 and RPMI 2650 cells to IFNγ for 48 h. Actin was used as internal control for loading and transfer. b Spectrofluorometric analysis shows the ability of 1-methyl-d-tryptophan (1-MT) to block IFNγ-induced IDO activity in both CLS-354 and RPMI 2650 cells. Cells were grown with medium alone or medium containing IFNγ in the absence or presence of 1-MT for 48 h. The supernatant was collected, and residual tryptophan was converted to norharman. Norharman levels were measured by a spectrofluorometer using 360 and 640 nm as excitation and emission wavelengths, respectively. The values are expressed as the mean ± SD of three independent experiments performed in duplicate. The student’s t-test was used for analysis. c Western blot analysis demonstrates the inhibition of INFγ-induced activation of IDO results in the rescue of IFNγ-induced suppression of HO-1 protein. Actin was used as internal control for loading and transfer. d Flow cytometry analysis of IFNγ-induced accumulation of the reactive oxygen species (ROS) using DHR 123 in RPMI 2650 and CLS-354 cells following the knockdown of IDO by its specific siRNA or the inhibition of IDO enzyme activity by 1-MT. ROS generation was measured by flow cytometry using dihydrorhodamine (DHR 123). The values are expressed as the mean ± SD of three independent experiments performed in duplicate. The Student’s t test was used for analysis