SENEX inhibits adhesion molecule expression and permeability. HUVECs were infected with SENEX and EV adenovirus for 24 hours. They were treated with 5 ng/mL TNFα in normal HUVEC medium for 5 hours and then stained for surface expression of E-selectin (A) or VCAM1 (C) (right panels); bar = 220 μm. GFP photos of the area photographed for adhesion molecule expression is given in the left-hand panels. The cells were also permeabilized and stained for intracellular E-selectin (B) and VCAM1 (D); bar = 220 μm. (E) Senescent and nonsenescent cells were analyzed for cell-surface expression of E-selectin and VCAM1. The mean pixel intensity per cell was measured using ImageJ for senescent cells (■) and for nonsenescent cells (□). The E-selectin data are a representative of the mean ± SEM of 25 senescent cells and nonsenescent cells from 2 HUVEC lines. ***P < .001 compared with nonsenescent cells. The VCAM1 data are a representative of the mean ± SEM of 14 senescent cells and the corresponding area of nonsenescent cells from 2 HUVEC lines. ***P < .001 compared with nonsenescent cells. (F) EV- and SENEX-infected cells were left for 5 days and then were either stimulated or unstimulated with 5 ng/mL TNFα for 4 hours, then stained for VCAM1 expression. Cells were analyzed for VCAM1 expression. No TNFα: purple line = EV, blue line = SENEX; TNFα stimulated: green line = EV, red line = SENEX cells. This is a representative of 3 experiments. (G) E-selectin expression performed as for panel F. (H) HUVEC monolayers seeded on a transwell membrane were infected with EV- (□) or SENEX (■)–expressing adenovirus. Passage of FITC-dextran through the membrane in response to thrombin was tested 48 hours later. This is a representative experiment of 3 showing the average of 4 transwells per condition ± SEM. *P < .05 compared with EV group with thrombin.