Fig. 6

β-catenin Y142 phosphorylation accounts for AGE-induced β-catenin transcriptional activity and angiogenesis. A After transfection with β-catenin WT or Y142F-overexpression adenovirus for 48 h, HUVECs were stimulated by AGEs (100 μg/mL) for 8 h. Proteins of the nuclear and whole cell fractions were isolated and then β-catenin protein level was detected by western blot, respectively. n = 6. NL indicates nuclear lysate; WCL indicates whole cell lysate. B-D 293T cells were co-transfected with β-catenin WT or Y142F-overexpression adenovirus and TCF/LEF response element (RE) (B), KDR promoter (C) or HDAC9 promoter (D) luciferase constructs followed by AGEs stimulation (100 μg/ml). The Dual-Luciferase Reporter Assay was conducted to detected luciferase reporter activities, and luminescence was normalized to Renilla. n = 5–6. E–G After transfection with β-catenin WT or Y142F-overexpression adenovirus for 48 h, HUVECs were stimulated with AGEs (100 μg/ml) for 24 h followed by the CCK8 assay (E), Transwell assay (F), and tube formation assay (G) to evaluate the OD value, migrated cells number and tube length, branching points, respectively. n = 3 to 6, scale bar indicates 100 or 200 μm. H Matrigel containing HUVEC transfected Ad-WT or Ad-Y142F in the presence or absence of AGEs (100 μg/mL) treatment was injected subcutaneously into mouse. A week later, plugs were harvested. Ulex europaeus agglutinin I (UEAI, Red) was used to stain HUVECs and HUVECs-formed blood vessels were measured. n = 3, scale bar indicates 100 μm. Ad indicates adenovirus overexpressing the corresponding protein. I Aortic rings were transfected with Ad-WT or Ad-Y142F, treated with or without AGEs (100 μg/mL) for 6 days and then stained with isolectin B4 (IB4; green). The mean number of sprout (indicated by red dot) from aortic ring was counted. n = 5, scale bar indicates 100 μm. Data are shown as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001