Figure 2

Functional correlation between miR-29a levels and HIV replication. (A) U1 cells and J1.1 cells were transfected with the pMIR-Report-Nef3′UTR reporter plasmid or the control pMIR-Report plasmid together with plasmid pRLTK. After 48 hr the cells were activated with PMA and the cell lysates quantified for luciferase activity as described in Materials and Methods. The luciferase activity in PMA activated cells is shown relative to unactivated cells after two normalizations - one with the Renilla luciferase activity to control for transfection efficiency and the other with luciferase activity observed with the control plasmid. Data represents three independent experiments; *p < 0.05. (B) J1.1 cells were transfected with the miR-29a-EGFP or EGFP expression vector. After 48 hr the cells were activated with PMA (or not) and HIV-1 production in culture supernatants was measured using a quantitative p24 ELISA. Transfection efficiency was around 30-40%. Data are shown as ng/ml of p24 and represent three independent experiments; the p-values for different sets are indicated. (C) U1 cells were transfected with a miR-29a specific LNA or a control LNA. After 48 hr HIV-1 production in culture supernatants was measured using a quantitative p24 ELISA. Data are shown relative to the EGFP control and represent three independent experiments; *p < 0.05. The western blot shows intracellular levels of Gag for the same cells; Actin was used as a loading control.