SENEX overexpression induces senescence characteristics. (A) HUVECs were infected with EV (i) or SENEX (ii) containing adenovirus. After 72 hours cells were stained for acidic β-galactosidase. Bars = 220 μm. (B) HUVECs were infected with SENEX containing adenovirus. The cells were stained with DAPI every 24 hours for the next 3 days. Top panel shows the cells at 24 hours with the GFP field with cells exhibiting polyploidy marked with an arrow. DAPI stain is shown in the bottom panel. Bar = 100 μm. (C) SENEX overexpression induces polyploidy. HUVECs, infected with EV (□) or SENEX (■) containing adenovirus, were assessed for polyploidy. At least 1000 cells were counted each day in each group for each of 3 individual cell lines and are presented as a percentage of the senescent cells. The mean ± SEM is shown. **P < .01 and ***P < .001 compared with EV. (D) SENEX overexpression inhibits EC proliferation. HUVECs, infected with EV (□) or SENEX (■) containing adenovirus, were assessed for cell proliferation after 4 days using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. OD_490nm for cells at day 0 and day 4 are given. Results are the mean ± SEM of a pool of 3 experiments where each experiment used a different HUVEC line, and each group in each experiment was done in 4 replicates. *P < .05 compared with EV on day 4. (E) SENEX overexpressing cell lysate (from adenovirus infected cells) was used for Western blotting with eNOS antibodies. β-actin was used as a loading control. This is a representative of 3 experiments.