Figure 5.

MMP-14 is a downstream target of endothelial SIRT1 inhibition. (A) Representative immunoblot using protein lysates isolated from primary HUVECs treated with 50 µM of a specific SIRT1 inhibitor (SIRT1 inhibitor III) for 48 hours. The inhibition of SIRT1 results in a profound decrease in MMP-14 expression levels. HUVECs cultured without addition of SIRT1 inhibitor served as a control. Cell lysates are separated by SDS-PAGE and analyzed by immunoblot using appropriate antibodies. β-Tubulin serves as a loading control. n=5. (B) A broad bright “halo” of proteolytically degraded matrigel, a functional manifestation of MMP enzymatic activity, is consistently seen around individual cultured HUVECs. When cells are cultured in the presence of different concentrations of SIRT1 inhibitor, the halo phenomenon exhibits dose-dependent attenuation or total disappearance. The lower panel depicts the results of line-scan analysis of halo density, whereas the right panels summarize quantitative analyses of capillary length, halo intensity, and integrated intensity. n=30 cells per group. Data are the mean ± SEM. *P<0.05.