Figure 2. Tat increases miR-222 levels in a NFkB-dependent manner. (A) HeLa cells were transfected with the empty vector (C), Flag-Tat or Flag-Tat CA expression constructs. Forty-eight hours post-transfection, total RNA was collected, and expression of miR-222 was analyzed by qRT-PCR. As a control, we checked the expression of MIP-1a, a NFkB-dependent gene that was shown to be sensitive to Tat.¹² Results shown represent the average of three independent experiments, all performed in triplicate. (B) ChIP assay of chromatin isolated from HeLa cells transfected with empty plasmid (C), Flag Tat, Flag-TatCA, or Flag-Tat plus pCMV-IkBam plasmids (Flag-Tat+IkBa), and immunoprecipitated by anti-Flag or control IgGs, followed by qPCR analysis with primers targeted either to miR-222 enhancer region, or to a sequence on chromosome 1 used as the negative control,¹¹ or to Mip-1a promoter used as positive control.¹² The data show occupancy relative to control IgGs and represent mean ± SD of three independent experiments. (C) Nuclear protein extracts from HeLa cells transfected with empty plasmid (C), Flag Tat, Flag-Tat plus pCMV-IkBam plasmids (Flag-Tat+IkBa), were harvested and p65 NFκB activity was measured by transAM p65 protein kit. *, P < 0.05; **, P < 0.01 by Student's t test