Fig. 3: overexpression of miR-484 and miR-744 modulates Dengue virus (DENV) replication. (A) Vero cells were transfected with pEZX-mR03-miR-484, pEZX-mR03-miR-744, or the empty vector pEZX-mR03 (scramble) and after 24 h, challenged independently with each of the four DENV serotypes at an MOI of 3; (B) quantification of DENV RNA copy number in the supernatants by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). (C, D) DENV-2 NS1 expression by western blotting for each condition evaluated and the band intensities quantified by densitometry, respectively. Ponceau red was used as the loading control for western blotting, as previously reported (Romero-Calvo et al. 2010, Gilda & Gomes 2013, Rivero-Gutiérrez et al. 2014); (E) Vero cells were first challenged independently with each DENV serotype and then transfected with pEZX-mR03-miR-484, pEZX-mR03-miR-744, or the empty vector pEZX-mR03. The percentage of infected cells was evaluated at 72 hpi by flow cytometry. The data are expressed as the percentage of infected Vero cells compared with those in the scrambled infected Vero cells, defined as 100% infection. Results are shown as Median and Range (two way ANOVA, p < 0.005); (F) Quantification of DENV RNA copy number in the supernatants by RT-qPCR. (G, H) DENV-2 NS1 expression by western blotting for each condition evaluated and band intensities quantified by densitometry, respectively. Ponceau red was used as the loading control for western blotting, as previously reported (Romero-Calvo et al. 2010, Gilda & Gomes 2013, Rivero-Gutiérrez et al. 2014). The data are shown as Median and Range (two way ANOVA). Three independent replicates were performed for each experiment. (*) Statistically significant difference compared to the control (p < 0.05).