Figure 4. Levels of miR-294-3p, miR-721, Nos2 mRNA, NOS2 and NO in BMDMs infected with La-WT, La-arg⁻ or La-arg⁻ + ARG L. (L.) amazonensis.

BMDMs (1 × 10⁶) were infected with La-WT (red) or La-arg⁻ (blue) or La-arg⁻ + ARG (orange) L. (L.) amazonensis (MOI 5:1). After 4, 24 and 48 h of infection, miR-294-3p (A), miR-721 (B) and Nos2 mRNA levels were quantified via qPCR (C), NOS2 protein levels was quantified via in-cell western blotting (D) and the average of fluorescence intensity reflecting NO production (E) was determined by flow cytometry analysis of DAF-FM staining and values was normalized by uninfected macrophages (100%). Each bar or dot represents the average ± SEM of the values obtained in 3 independent experiments (n = 4–6). Statistical significance was determined based on two-tailed Student’s t test. *p < 0.05, compared to uninfected macrophages (0 hours of infection). **p < 0.05, La-arg⁻ or La-arg⁻ + ARG compared to La-WT. ***p < 0.05, La-WT or La-arg⁻ + ARG compared to La-arg⁻.