Fig. 5

LP-induced eNOS signaling mediates phosphorylation of LKB1/AMPK. Representative western blot from a (A) dose-response and (B) time course experiment analyzed in cells stimulated with LP for 30 s and 60 s. Cell lysates were used to determine the phosphorylation of AMPK and LKB1 by western blot using antibodies specific to the phosphorylated protein. The total AMPK and LKB1 levels were also assessed as controls for loading. The band intensities were measured and are represented as a graph. ***, P < 0.001. LKB1/AMPK signaling regulates LP-induced angiogenesis and migration in the upstream pathway. HUVECs were transfected with AMPK-siRNA (100 pmol) or control siRNA for 24 h and then treated with LP for 60 s. C After 24 h, cell lysates were analyzed by western blotting using antibodies against p-AMPK, T-AMPK, p-eNOS, and T-eNOS. D Protein expression of VE-cadherin and ECM proteins p-FAK(Y397)), FAK, p-Src(Y418), and Src, as assessed by western blotting. E Angiogenic activity of AMPK siRNA-transfected HUVECs determined using a tube formation assay; scale bar = 1000 μm. Bar graph showing quantification of tube formation. N = 5, ***P < 0.001. F Migration of AMPK siRNA-transfected HUVECs in a wound migration assay. Bar graph showing quantification of cell migration. ***P < 0.001; scale bar = 1000 mM. G, H Immunocytochemical assays for VE-cadherin and p-FAK. G The expression of VE-cadherin was decreased upon LP treatment, and the decrease in VE-cadherin expression was mitigated in cells transfected with AMPK siRNA; scale bar = 20 μm (H) Expression of p-FAK was attenuated in AMPK siRNA transfected cells. The outlined areas are enlarged in the side panels. Scale bar = 30 μm