SENEX expression is regulated by H₂O₂ and TNFα. (A) HUVECs were stimulated with 100μM H₂O₂ for 2 hours and then placed in normal HUVEC medium for 24 hours (ii) or kept in normal HUVEC medium for 24 hours (i). Cells were then stained for β galactosidase activity. Bar = 100 μm. (B) HUVECs were stimulated as in panel A and then placed in normal HUVEC medium for 24 hours. SENEX expression was measured by Western blot with β-actin used as a loading control. This is a representative of 3 experiments. (C) HUVECs were treated with 500μM H₂O₂ for 6 hours and then were assessed for apoptosis using the caspase 3 activity assay. The mean ± SEM of 6 replicates from 3 lines of HUVECs is shown. ***P < .001 compared with no H₂O₂ treatment_. (D) HUVECs were treated as in panel C. SENEX expression was measured by Western blot with β-actin as a loading control. This is a representative of 3 experiments. (E) Cells were treated with 10 ng/mL of TNFα in normal HUVEC medium for 24 hours. Protein lysates were harvested at 6 and 24 hours and the SENEX protein levels were measured using Western blotting. β-actin was used as a loading control. This is a representative of 3 experiments. (F) HUVECs were infected with SENEX (■) and EV (□) adenovirus for 24 hours. They were treated with 10 ng/mL TNFα in normal HUVEC medium for 24 hours or cultured in serum-free HUVEC medium for 24 hours or left untreated. Protein lysates were harvested and apoptosis measured using a caspase 3 activity assay. The mean ± SEM of 6 replicates from 3 lines of HUVECs is shown. *P < .05 and ***P < .001 compared with EV.