Surface Expression of Junctional Proteins Increases upon Monocyte Maturation.

<p>The surface expression of JAM-A, ALCAM, CD99, and PECAM-1 was analyzed by flow cytometry on freshly isolated, “Day 0” and “Day 3” monocytes. (<b>A</b>) FACS data represented as histograms show the surface junctional protein expression from one representative HIV(-) individual. The mean fluorescent intensity (MFI) of each protein, or the MFI obtained with each isotype matched negative control antibody, is indicated on “Day 0” and “Day 3”. There was an increase in each junctional protein as the monocytes matured from “Day 0” (blue) to “Day 3” (green). (<b>B</b>) After subtracting the contribution of the isotype matched negative control antibodies, the MFI of JAM-A (red), ALCAM (blue), CD99 (yellow), and PECAM-1 (green) were analyzed on monocytes from 30 different HIV(-) individuals on “Day 0” (circles) and “Day 3” (squares). The extent to which each junctional protein increased after nonadherent culture for 3 days varied among donors. (<b>C</b>) The fold increase in the MFI of each junctional protein on “Day 3” monocytes (shaded bars) from 30 individual HIV(-) donors relative to “Day 0” (white bars, set to 1) was determined. (<b>D</b>) qRT-PCR was performed to determine the mRNA expression of junctional proteins. RNA was isolated from “Day 0” and “Day 3” monocytes from 4 independent donors. The fold change on “Day 3” (solid bars) relative to “Day 0” (white bars, set to 1) of JAM-A (red), ALCAM (blue), CD99 (yellow), and PECAM-1 (green) normalized to β-Actin was determined. Data are represented as mean ± standard error of the mean. Significance was determined using Wilcoxon Signed Rank test or a Two-Tailed Paired T test. ^*p<0.05**p<0.01. ALCAM was not significant due to high inter-donor variability, despite the large increases in mRNA expression (p=0.1).