Figure 4. Mito-Esc restores H₂O₂-induced inhibition of AMPKα and eNOS phosphorylation in HAEC.

(a) HAECs were pretreated with either Mito-Esc (2.5 μM) or esculetin (2.5 μM) for 2 h before H₂O₂ (500 μM) was added for 8 h and nitric oxide levels were measured by DAF-2DA fluorescence as described in methods. (b) Same as a, except that nitrite levels were measured. (c–f) HAEC were treated with various conditions as indicated for 8 h after which AMPKα, eNOS and phospho-eNOS (Ser-1177) protein levels were measured by western blot analysis. (g) HAEC were pretreated with esculetin, Mito-Esc, L-NAME (2 mM) for 2 h before the addition of Ang-II or H₂O₂ for 24 h and cell viability was measured by trypan blue assay. (h–j) Same as c, e, f except that, phospho-AMPKα (Thr-172) and AMPKα, protein levels were measured by immuno blotting. AMPKα (k) HAEC were treated with Mito-Esc in presence or absence of Compound c for 8 h and eNOS and p-eNOS (Ser-1177) protein levels were measured by Western blot analysis. The data shown represents from three independent experiments *significantly different (p < 0.05) compared to untreated conditions. ^#Significantly different (p < 0.05) compared to Ang-II or H₂O₂ treated condition. ^$Significantly different (p < 0.05) compared to Esculetin + H₂O₂ treated condition.