Figure 2
vGPCR activates Wnt/β-catenin signaling in endothelial cells. (A) Total RNA was isolated from BABE- and BABE-vGPCR-expressing HUVECs and analyzed with Human Wnt signaling pathway RT2 Profiler PCR array (SABiosciences, Frederick, MD). Genes that displayed greater than 3-fold change over control were considered significant. (B) HUVECs expressing vGPCR or BABE vector were serum starved for 16 hr and fractionated into cytosolic and nuclear components. Protein fractions were analyzed by Western blot with an anti-β-catenin antibody. Anti-γ-tubulin, and anti-H3 Western blots were included as controls for the purity of the fractionation procedure. β-actin served as a loading control. The densitometry data presented below the bands are arbitrary units normalized to the respective loading control (α-tubulin or histone H3). (C) HUVECs stably transduced with BABE or BABE-vGPCR were seeded on glass coverslips and stained with rabbit polyclonal antibody to β-catenin followed by AlexaFluor 555-conjugated anti-rabbit IgG. Nuclei were counterstained with DAPI. 40 X magnification. (D) HUVECs expressing vGPCR or control vector (BABE) were transiently transfected with TOPflash or FOPflash firefly luciferase-expressing plasmids. pRL-TK plasmid expressing Renilla luciferase was co-transfected as a control. Forty-eight hr after transfection, the cells were collected in lysis buffer and luciferase expression in cell lysates was measured and normalized to Renilla activity. Data are presented as the mean of results from two independent experiments each performed with triplicate transfections. ***p < 0.001. (E) Cyclin D1, MMP-9 and VEGFA mRNA levels in BABE-vGPCR- and BABE-expressing HUVECs were analyzed by qRT-PCR in triplicate. Each sample was normalized to the level of 36B4 expression. The data are presented as fold change (mean +/− SEM) relative to BABE control sample. *p < 0.05; ***p < 0.001.