FIGURE 6.

Fstl1-stimulated endothelial cell responses are dependent on Akt signaling. A, Fstl1-stimulated signaling in endothelial cells. HUVECs were transduced with Ad-Fstl1 and Ad-β-gal for 8 h followed by 24 h of incubation with serum-free media. Changes in the phosphorylation of eNOS (P-eNOS), Akt (P-Akt), GSK (P-GSK), and ERK (P-ERK) following Ad-Fstl1 treatment were determined by Western blot analysis. Representative blots are shown. Relative phosphorylation levels of eNOS and Akt were quantified (n = 6) by using ImageJ. Immunoblots were normalized to total loaded protein. B, role of Akt in regulation of Fstl1-induced signaling. HUVECs were infected with adenoviral constructs encoding dominant-negative Akt1 (Ad-dnAkt) or Ad-β-gal at an m.o.i. of 10 along with Ad-Fstl1 or Ad-β-gal at an m.o.i. of 10 for 8 h, followed by serum deprivation for 24 h. Phosphorylation of eNOS (P-eNOS) and Akt (P-Akt) were determined by Western blot analysis. Representative blots are shown from four independent experiments. C and D, contribution of Akt to Fstl1-mediated cellular responses. HUVECs were transduced with Ad-dnAkt or Ad-β-gal along with Ad-Fstl1 or Ad-β-gal for 8 h. After 24 h of serum deprivation, Matrigel (C) or modified Boyden chamber assays (D) were performed. E, involvement of Akt in Fstl1-induced endothelial cell survival. After transduction with Ad-dnAkt or Ad-β-gal along with Ad-Fstl1 or Ad-β-gal for 8 h, cells were incubated in serum-free media. Nucleosome fragmentation was assessed by enzyme-linked immunosorbent assay. Results are shown as the mean ± S.E. (n = 6-8). Results are expressed relative to the values compared with control. ^*, p < 0.01.