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. 2013 Aug 5;8(8):e71044. doi: 10.1371/journal.pone.0071044

Figure 5. Inhibition of iNOS expression and NO production by tryptophan deficiency in IFN-γ+LPS stimulated RAW264.7 cells.

Figure 5

(A) Q-PCR analysis of iNOS mRNA expression. Raw264.7 cells were pre-incubated in RPMI, tryptophan-deficient medium (Trp-D), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan or RPMI supplemented with increasing concentrations of kynurenine (10, 25, and 50 µg/ml) for an hour. Cells were then stimulated with IFN-γ+LPS for 12 hours. Cells were collected, and iNOS expression was determined by Q-PCR after RNA extraction and cDNA synthesis. GAPDH was used as the reference gene. The relative expression of iNOS mRNA is evaluated by Q-PCR in Raw264.7 cells cultured and treated in different conditions. The iNOS expression was normalized to the GAPDH expression level (B) Raw264.7 cells were cultured and stimulated for 24 hours as described above. The concentration of released nitric oxide was evaluated in the cell supernatant by the Griess method. Data is mean±SEM of four independent experiments (**P-value<0.01, n = 4).