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. 2015 Feb 10;12(2):123–135. doi: 10.1080/15476286.2015.1014759

Figure 1.

Figure 1.

RRE inhibits miRNA-mediated RNAi activity in the cell. (A) Comparison of TAR and RRE expression in transfected HeLa cells and in HIV-infected Jurkat lymphocytes. HeLa cells (lanes 1–9) were transfected with 1 μg of pcDNA3, pcDNA3-TAR, pcDNA3-RRE, pSIREN, pSIREN-TAR, pSIREN-RRE or pNL4–3, as indicated. Jurkat cells (lanes 10–12) were infected with pNL4–3 and harvested at days 0, 7 and 13 of infection. Expression of TAR, RRE and GAPDH were detected by RT-PCR. (B) Schematic representation of the EGFP and Renilla Luciferase (RL) assays to measure the RNAi activity mediated by let7. (C) RRE prevents let7-mediated inhibition of EGFP expression. HeLa cells were cotransfected with 0.4 μg pEGFP or pEGFP-clet7 and with 1μg of pcDNA3, pcDNA3-TAR or pcDNA3-RRE, or 1 μg pCMV1-Tat or pCMV-Rev as indicated. Effects on knockdown of EGFP by miRNA let7 were observed by immunoblot (left), and quantified by densitometry analysis. Histogram (right) shows the average RNAi activity (EGFP knockdown) over 3 independent experiments ± SEM. (D) TAR and RRE prevent let7-mediated inhibition of RL expression. HeLa cells were transfected with 5 ng of pRL or pRL-clet7 and with 0.25 μg of pcDNA3, pcDNA3-TAR, or pcDNA3-RRE, or 0.25 μg pCMV1-Tat or pCMV-Rev as indicated. (E) TAR and RRE prevent miR17-mediated inhibition of RL expression. HEK 293T cells were transfected with 5 ng of pRL or pRL-cmiR17 and with 0.25 μg of pcDNA3, pcDNA3-TAR, or pcDNA3-RRE as indicated. (D and E) Luciferase signals were measured using a luminometer. Signals were normalized to the cotransfected pCMV-firefly luciferase (FL). RNAi activity (Luc knockdown) is calculated as the ratio of RL/FL to RL-let7/FL or to RL-miR17/FL, and displayed as per cent of RNAi activity with the empty plasmid. Histogram shows the average of 3 independent experiments ± SEM.