Function of miR-320b in endothelial cells. (A) Reporter gene signal from HEK293 cells transfected with 10 ng of a reporter plasmid containing the 3′-UTR of ICAM-1 and 20 or 100nM precursor miRNA corresponding to miR-22 or miR-320b and 10 ng of a reporter plasmid containing the 3′-UTR of ICAM-1. The reporter signal (Firefly luciferase) was normalized to Renilla luciferase to account for differences in transfection efficiency and expressed relative to the mean of the cells transfected with plasmid alone. Data come from 3 separate experiments with each sample run in triplicates. **P < .01; ***P < .001. (B) The level of ICAM-1 expression in HMEC-1 cells cultured for 24 hours in the presence of 20% platelet releasate. Gene expression was measured by qRT-PCR and is presented relative to the expression of cyclophilin A and normalized against the mean of the untreated control samples. Data from 3 separate experiments using triplicates are presented. *P < .05. (C) The levels of ICAM-1 in HMEC-1 cells upon treatment with 100nM scrambled anti-miRNA (anti-miR-Scr), anti-miR-22, or -320b in the presence or absence of 20% platelet releasate. Expression data are handled as mentioned previously (n = 6). *P < .05; ***P < .001 comparing cells treated with anti-miR-scr and anti-miR in the absence of platelet releasate; ##P < .01, comparing cells treated with anti-miR in the absence of releasate with cells treated with a combination of anti-miR and releasate. (D) Representative FACS plot of HMEC-1 cells transfected with 100nM pre-miR-320b (black line) or scrambled control pre-miRNA (gray line) and stained with a FITC-conjugated monoclonal antibody to ICAM-1. FL-1 fluorescence intensity reflects the surface expression of ICAM-1. (E) Surface expression of ICAM-1 in HMEC-1 (n = 3) transfected with 100nM pre-miR-320b or scrambled control pre-miRNA assessed with flow cytometry. ICAM-1 levels are expressed as the mean fluorescence intensity (MFI).***P < .001.