Supplementary Figure 4: CCR5AS silencing inhibits CCR5 expression in Hut-78 cells by reducing CCR5 mRNA stability.

a, Hut-78 cells were transfected with 300 nm siRNA targeting CCR5AS (siLnc1) or control siRNA (siCon1). CCR5 mRNA was measured 24 hours post-transfection. Cells transfected with siLnc1 showed lower expression as compared to the controls. b, Cell surface expression of CCR5 on Hut-78 cells was measured 48 hours post-transfection. The cells transfected with siLnc1 (red curve) showed lower cell surface expression as compared to siCon1 transfected cells (blue curve). The gray curve depicts isotype controls. 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 three comparable experiments performed is shown. The mean ± SE (n = 3) 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. c, CCR5 mRNA RNA decay was determined by 5-Ethylene uridine (EU) pulse-labeling of RNA using the Click-iT Nascent RNA Capture Kit. Hut-78 cells were transfected with siCon1 or siLnc1 and pulsed with EU. Eighteen hours after EU-pulsing, the cells were washed, supplemented with fresh growth medium and harvested at one hour intervals for 4 hours. Total RNA was isolated from cells and quantitated. The EU-labeled RNA was biotinylated, precipitated, and captured using streptavidin coated magnetic beads as per the manufacturer’s protocol. The RNA captured on beads was used for cDNA synthesis and qPCR analysis. Data are represented as relative expression levels of CCR5 mRNA in the siCon1 or siLnc1 transfected cells. The mRNA half-life (50% mRNA remaining, t_1/2) for the CCR5 mRNA in siCon1 (3h) was higher as compared to the siLnc1 treated cells (2.2h). Data represent mean ± SEM. One of two comparable experiments performed is shown. Paired t test was used for statistical comparisons and one tailed p value is indicated.