Figure 5

Expression of DNMTs and DNMT-regulating miRNAs in Tat-positive vs. Tat-negative cells. DNMT1 and DNMT3a/b-regulating microRNA expression was checked by RT-qPCR in Tat-positive vs. Tat-negative cells following transient transfections and exposure to recombinant protein (a). Down-regulation of all miRNAs is observed in Tat-positive cells. The graph is representative of three different RT-qPCR experiments. Error bars represent standard deviation between duplicates. (b-d) Modulation of DNMT-regulating miRNAs was achieved by transient transfections of either mimics or antagomirs of the endogenous miRNAs at different concentration, and the effect on the expression of their respective miRNAs was monitored by RT-qPCR. DNMT1-regulating miRNAs (hsa-miR130a and hsa-miR152) are reported in (b-c), whereas DNMT3a/b regulating miRNAs (hsa-miR29) are shown in (d). (e-f) Relative expression of DNMT1 (e), DNMT3a/b (f) was then evaluated following the ectopic modulation of these miRNAs. Up-regulation of the specific miRNAs results in the down-regulation of DNMTs, whereas increased expression of DNMTs is observed following miRNA inhibition. The graph is representative of three different RT-qPCR experiments (p < 0.05). Error bars represent standard deviation between duplicates. (g-h): WB for DNMT1 and DNMT3a of cells transfected with hsa-miR152 (for DNMT1) and hsa-miR29 (for DNMT3a) mimics and inhibitors. NC: Mimic negative control; M: Either hsa-miR152 or hsa-miR29 mimic; NCI: Inhibitor negative control; I: Either hsa-miR152 or hsa-miR29 inhibitor. None of the tested antibodies for DNMT3b was suitable for WB analysis. Densitometric analysis results are reported.