miRs-29a, -29b, -9 and -146a reduce expression of a luciferase reporter and target predicted binding sites in the 3′ UTR of SIV. (A) SIV nef/U3-R was cloned into psiCHECK2 plasmid (Promega). 293T cells were co-transfection with 50 nM or 100 nM of each miRNA or scrambled mimic and 100 ng of either psiCHECK+SIV or psiCHECK alone. Control samples were transfected with either psiCHECK or psiCHECK+SIV, and no miRNA mimics. Cells were harvested 24 hours after transfection. Data shown is an average of four experiments and represents miRNA effect on luciferase expression for psiCHECK+SIV over psiCHECK only. (B) WT and mutant biotin-labeled oligos corresponding to SIV RNA sequences containing predicted miRNA binding sites. WT oligo is show on top and mutant on bottom. Boxed bases on WT oligo denote miRNA seed binding site in SIV sequence. Mutant oligo highlighted bases denote the change in seed-binding region. Annotation is based on SIV sequence AY033146. (C) 100 pmol of individual WT or mt oligos were transfected into HeLa cells and lysates collected 24 hours after transfection. Oligos were pulled down using Streptavidin beads and assayed for binding to endogenous cellular miRNAs using Taqman RT-qPCR. Percent input was calculated for each sample (see methods) and data presented as (% input mt )/(% input WT) with % of input of WT set to 100%. In each experiment, the value of the oligo which pulled down the highest percentage of individual endogenous miRNA was set to 100% and binding by other oligos was compared to this value. Data shown is an average of 4 experiments. Statistics represent the comparison of percent of miRNA bound to WT oligo compared to percent of miRNA pulled down by corresponding mt oligo and are reported as a two-tailed t test assuming unequal variance.