Effect of PI3K inhibition on Wnt/β-catenin activation by vGPCR. (A) HUVECs expressing vGPCR were transiently transfected with TOPflash and pRL-TK plasmids. Twenty four hr after transfection, the cells were treated with 25 μM LY294002 or vehicle control for an additional 24 hr. Cell lysates were collected and analyzed for luciferase expression as described in Figure 3A. (B) HUVECs expressing vGPCR were pretreated with 25 μM LY294002 or vehicle control for 12 hr and plated in 96-well plate coated with Matrigel in the presence of LY294002. (C) The number of branch points was determined as in Figure 3B. ***p < 0.001. (D) Cytotoxicity of increasing doses of LY294002 was determined by MTT assay *p < 0.05. (E) Cyclin D1, VEGFA and MMP-9 mRNA expression was measured by qRT-PCR in control (BABE), vGPCR- expressing cells, and vGPCR-expressing cells that were treated with 50 μM LY294002 for 24 hr. The data are presented as fold change (mean +/− SEM) relative to control (BABE) sample. *p < 0.05; **p < 0.01.