Figure 2.

Effect of SIRT1 on cellular responses in endothelial cells. (A,B) Representative micrographs and statistical summary of three-dimensional in vitro angiogenesis assays with collagen gel-embedded spheroids generated from SIRT1 or scrambled siRNA-transfected endothelial cells. (C,D) Representative micrographs and statistical summary of in vitro Matrigel assays with SIRT1 or scrambled siRNA-transfected endothelial cells. (E) HUVECs were transfected with a SIRT1-specific siRNA or a nonrelated scrambled control. Cell lysates were subjected to Western blotting using antibodies against SIRT1. Antibodies recognizing Foxo1, Akt, or tubulin were used as a loading control. (F–I) Statistical summary of the effects of SIRT1 gene silencing (white bars) on migration, adhesion, proliferation, and apoptosis compared with scrambled siRNA-transfected controls (black bars). The VEGF and bFGF concentrations were 50 ng/mL and 30 ng/mL, respectively. (J) HUVECs were transfected with wild-type (wt) SIRT1, a deacetylation-defective SIRT1 mutant (H363Y), or mock control (pcDNA3). Cells were lysed after 24 h and subjected to Western blot analysis with antibodies against SIRT1 and Myc. (K) Statistical summary of the cumulative sprout length in pcDNA-, SIRT1 wild-type-, or SIRT1 H363Y-transfected endothelial spheroids. (L) Statistical summary of endothelial migration toward the chemoattractant VEGF (50 ng/mL) in pcDNA-, SIRT1 wild-type-, or SIRT1 363Y-transfected cells.