Fig. S5.

Metacyclic L. major infections reduce PEM iNOS levels and nitrite production when measured after 24 but not 72 h. (A) Schematic of experimental approach for B–F. PEMs were infected with metacyclic stage L. major expressing GFP (time 0) and stimulated with IFN-γ and LPS either 2 h after infection (short infection) or 72 h after infection (long infection). After 24 h, nitrite was determined in cell supernatants, and cells were processed for confocal microscopy. Controls included uninfected/unstimulated and uninfected/stimulated. (B) Nitrite levels in PEM supernatants are decreased after 24 but not 72 h of infection. (C) Confocal microscopy of PEMs after the “short infection” protocol. Parasites are shown in green (GFP), anti-iNOS reactivity in red, and anti-GAPDH reactivity in blue (MΦ cytosol). Parasite-containing PEMs show reduced anti-iNOS intensity and thus appear “blue.” (Scale bar, 10 µm.) (D) Relative iNOS expression was determined from images acquired in the experiments shown in C by dividing the anti-iNOS fluorescence by anti-GAPDH fluorescence measured using image analysis software. Each data point represents one host cell. For infected samples, only cells containing parasites were selected for analysis. (E) Confocal microscopy of PEMs after the “long infection” protocol. Images were generated and labeled as described in C. (F) Relative iNOS expression of cells under long infection conditions, as described in D. Data, mean ± SEM; *P < 0.05; N.S., not significant (P > 0.05; ANOVA).