Fig 6. Inhibition of NOS activity during La-WT and La-arg^- amastigotes differentiation.

(A) L. (L.) amazonensis La-WT at early-stationary phase promastigotes (pro) were differentiated into amastigotes (ama), by changing the M199 medium to pH 5.5 and maintaining the culture at 34°C during 4 days, untreated (white) or treated with 10 mM of L-NAME (light gray) at day 0 or day of culture. Next, a total of 2x10⁶ cells was labeled with 1 μM DAF-FM and 1 ng/mL propidium iodide (PI) and analyzed using FACSCalibur (Becton Dickinson). B) Frequency of of DAF-FM⁺ cells. (C) Normalization of the percentage of DAF-FM⁺ cells in relation to La-WT-untreated (100%). (D) MFI of DAF-FM⁺ cells. (E) Normalization of the MFI of DAF-FM⁺ cells in relation La-WT-untreated (100%). (F) Number of amastigotes forms. (G) Normalization of number of amastigote forms in relation La-WT-untreated (100%). Each bar represents the mean of the values ±SEM (n = 3–9). These data were analyzed by Student’s t-test, and (*) p < 0.05. The data are representative of 3 independent experiments.