Figure 6.

GW0742-induced HUVEC tubulogenesis is SIRT1-dependent, but independent of any change in FOXO1 activation status (a) GW0742 (100 nM), but not VEGF-A (25 ng/ml), mediated tube formation was significantly reduced in the presence of the selective SIRT1 inhibitor, Ex-527 (1 µM), at 16 h. Data represent mean (±S.E.M) number of branches/field from n = 3. *p < 0.05 vs. untreated control; ^#p < 0.05; ^&p < 0.05 vs. Ex-527 control; as determined by two-way repeated measures ANOVA followed by Bonferroni’s post-comparison test. (b) FOXO1 acetylation status was not changed following treatment (1 h) with either VEGF-A (25 ng/ml) or GW0742 (100 nM). Data represents means (±S.E.M) from n = 3. For each n number, fluorescence intensity was analysed from 150 cells per treatment condition. (c) Densitometry analysis showing that FOXO1 phosphorylation is not significantly changed following treatment (1 h) with GW0742 (100 nM) (n = 3) but is significantly increased following treatment (1 h) with VEGF-A (25 ng/ml) (n = 2) compared to untreated control (n = 3). Data represent means (±S.E.M). *p < 0.05 vs. untreated control as determined by one-way ANOVA followed by Bonferroni’s post-comparison test. (d) Representative western blot showing phosphorylated FOXO1, total FOXO1 and β-actin loading control. Boxed areas indicate non-adjacent lanes on the original blot (see Supplementary Fig. S8).