Figure 5: Inhibition of miR-294 and miR-721 functions increased NO production and reduced the infectivity of L. (L.) amazonensis. | Scientific Reports

Figure 5: Inhibition of miR-294 and miR-721 functions increased NO production and reduced the infectivity of L. (L.) amazonensis.

From: Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism

Figure 5

BMDMs (5 × 105) were transiently transfected with the negative control, 30 or 100 nM of miR-294-5p (A–E) or miR-721 (F–J) inhibitors, or left non-transfected (untreated, black bars). After 24 h of incubation, the cells were co-cultivated with La-WT L. (L.) amazonensis (MOI 5:1) for 4 h, and the cultures were then washed. After 4 and 24 h of infection, the samples were analyzed for miR-294 (A), miR-721 (F) and Nos2 mRNA (B,G) levels via RT-qPCR of total RNA, for NOS2 expression (C,H) via in-cell western, for NO production (D,I) via flow cytometry DAF-FM fluorescence analysis, and for infectivity (E,J) via microscopy analysis, counting of the numbers of infected macrophages and amastigotes per macrophage (n = 1,000 macrophages/treatment). The values were normalized based on the average values of untreated infected macrophages. Each bar 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 negative control infected macrophages.

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