Angiogenic and transforming properties of vGPCR. (A) HUVECs were infected with the BABE retrovirus expressing vGPCR-GFP or a BABE-GFP control and imaged at 72 hr post transduction. (B) Cell migration was assayed using BD FluoroBlok cell culture inserts. BABE- and BABE-vGPCR-expressing HUVECs were loaded into the upper chamber of the inserts and allowed to migrate towards complete media for 12 hr. Migrated cells were stained with calcein AM and visualized by fluorescence microscopy. Average fluorescence intensity was determined using ImageJ software. Data are represented as the mean ± SEM (n = 3). **p < 0.001. (C) BABE- and BABE-vGPCR-expressing HUVECs were seeded in a 96-well plate pre-coated with growth factor-reduced Matrigel. Wells were examined for tubule-like structures after 6 hr and tube formation was quantified by counting the number of branch points (D). Columns represent the average number of branch points from two independent experiments performed in duplicate ± SE. *p < 0.05. (E) NIH3T3 cells stably transduced with BABE-vGPCR or BABE were mixed with high concentration, growth-factor-reduced Matrigel. Matrigel-cell suspension was injected subcutaneously into the left and right flanks of 6–8 weeks old athymic NCr-nu/nu mice. n (BABE) = 4; n (vGPCR) = 6. Plugs were retrieved, fixed and processed for hematoxylin-eosin staining. Macroscopic appearance of representative plugs containing BABE- (panel a) or BABE-vGPCR-expressing cells (panel b). Microscopic examination of hematoxylin-eosin stained sections from Matrigel plugs containing BABE- (panel c) or BABE-vGPCR-expressing cells (panel d). vGPCR plugs show an increase in formation of blood vessels containing red blood cells.