Supplementary Figure 3: CCR5AS enhances CCR5 mRNA and cell surface expression.
From: CCR5AS lncRNA variation differentially regulates CCR5, influencing HIV disease outcome
a, Peripheral blood CD4+ T cells were transfected with 300 nm siRNA targeting CCR5AS (siLnc1) or a control siRNA (siCon1). CCR5AS mRNA was measured 24 hours post-transfection. Cells transfected with siLnc1 showed 70–80% downregulation of CCR5AS mRNA as compared to the siCon1 treated cells. b, Cells transfected with siLnc2, which targets a region of CCR5AS distinct from that of siLinc1, showed 60–70% downregulation of CCR5AS mRNA as compared to siCon2 treated cells. c, Cells transfected with siLnc2 showed lower CCR5 mRNA expression as compared to the siCon2 transfected cells. d, Cell surface expression of CCR5 was measured 24 hours after siRNA transfection. The cells transfected with siLnc2 (red curve) showed lower CCR5 cell surface expression as compared to the siCon2 (blue curve) transfected cells. The open black curve depicts isotype control. Fold change in expression levels was calculated as the ratio of mean fluorescence intensity (MFI) of CCR5 vs isotype control. A histogram of one of 6 comparable experiments performed is shown for each silencing. The mean ± SE (n = 6) are depicted as horizontal and vertical bars for each group, respectively. Paired t test was used for statistical comparisons and two tailed p value is indicated. e, CD4+ T cells were transfected with the in vitro transcribed long form of CCR5AS [CCR5AS(L)] or scrambled RNA controls (ConRNA). CCR5 mRNA expression was measured 24 hours post-transfection. Cells transfected with CCR5AS(L) showed higher CCR5 mRNA levels as compared to the scrambled control. f, The cell surface expression of CCR5 in cells transfected with CCR5AS(L) (red curve) was higher as compared to the scrambled control (blue curve) transfected cells. The grey curve depicts isotype control. Fold change in expression levels was calculated as the ratio of mean fluorescence intensity (MFI) of CCR5 vs isotype control. A histogram of one of 6 comparable experiments performed is shown. Statistics are as described in (d).
