Figure 6. miR-294 and miR-721 bind to Nos2 mRNA and regulating NOS2 expression and L. (L.) amazonensis infectivity.
BMDMs (5 × 105) were plated into chamber slides overnight and transfected with negative control, 0.1 or 0.5 μM miScript Target Protector for miR-294/Nos2 or miR-721/Nos2 or negative control for 24 h. Then, the BMDMs were co-cultivated with La-WT L. (L.) amazonensis (MOI 5:1) for 4 h, and the cultures were washed. After 4 h and 24 h of infection, the samples were analyzed for NOS2 protein levels (A,D) via in-cell western, NO production (B,E) via flow cytometry DAF-FM fluorescence analysis, and for infectivity (C,F) 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 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.