Supplementary Figure 5: CCR5AS mediated regulation of CCR5 expression is independent of shared miRNA binding sites.

Hut-78 cells were transfected with in vitro transcribed CCR5AS or scrambled RNA controls and cell surface expression of CCR5 was measured 48h post-transfection. a, b, Cells transfected with the wild type long (L) form (a) or the short (S) form (b) of CCR5AS transcript showed higher CCR5 cell surface expression. c,d,e, Cells transfected with CCR5AS mutant transcripts with disruption in binding sites of miR-1224 (c), miR-197 (d), or both (e) showed higher CCR5 cell surface expression as compared to the respective controls. Surface expression of cells transfected with CCR5AS are shown as various colored curves whereas the scrambled RNA controls are represented by blue curves. The open black curves depict isotype control. A histogram of one of three comparable experiments performed is shown for each overexpression. f, Fold change in CCR5 expression levels was calculated as the ratio of mean fluorescence intensity (MFI) when staining with CCR5-specific antibody vs isotype control. g, Fold changes in CCR5 mRNA upon transfection with the various forms of the CCR5AS mRNA are shown. CCR5 mRNA levels were normalized to GAPDH mRNA and the mean ±SE (n=3) are depicted as horizontal and vertical bars for each group, respectively. CCR5AS RNA transfections were compared to those with Control RNA and an unpaired t test was used for statistical comparisons with two tailed p value indicated.