ADTRP post-transcriptional silencing decreases ADTRP and TFPI expression. (A) Total lysates of HUVECs transiently expressing nontargeting shRNA (control, lane 1) or ADTRP-shRNA (lane 2) were analyzed by SDS-PAGE and immunoblotting with rabbit anti-ADTRP, anti-TFPI IgGs, and mAb anti ß-actin. (B) Immunostaining for cell surface ADTRP and TFPI, and Cav-1 after permeabilization, on EA.hy926 cells transiently expressing nontargeting shRNA or ADTRP-shRNA. Bars: 10 μm. (C) Scatter-plot and statistical analysis (1-way ANOVA) of MFI for ADTRP, TFPI and Cav-1 in control EC (nontargeting shRNA) and EC expressing ADTRP-shRNA. MFI was measured on > 50 individual cells per experimental condition and expressed as arbitrary units (AU). Data are mean ± SEM. (D) Correlation analysis performed on individual cells, either control or ADTRP-shRNA EC, to analyze the relationship between ADTRP and TFPI fluorescence levels on the EC surface. r2 and slope deviation from 0 are highly significant for both experimental conditions. (E) ELISA on control ECs and ADTRP-shRNA cell monolayers (24-well plates) shows antigen levels for ADTRP and TFPI on the cell surface (S) and in permeabilized cells (P). Antigen values are expressed as ratios between the absorbance measured at 450 nm after developing the peroxidatic reaction with OPD, and the fluorescence intensity of DAPI used for normalization of cell numbers. Data are mean ± SEM; n = 6. ***,###: P < .001 versus control cells (unpaired t test).
