HLX causes strong inhibition of sprouting which is mediated to a significant part by UNC5B. HUVECs were infected with Ad.HLX, Ad.UNC5B, or Ad.con. The following day spheroids of infected and noninfected endothelial cells were generated as described in “Spheroid-based in vitro angiogenesis assay.” The spheroids were embedded into collagen gels and induced with VEGF or bFGF (25 ng/mL) or cultured without cytokine stimulation. After 24 hours, the spheroids were fixed and photographic images were taken for quantification. (A) Representative images of spheroids generated from Ad.HLX- or Ad.con-infected cells (10 MOI) and induced with VEGF (+) or left untreated (−) are displayed. (B) Quantification of inhibition of sprouting by overexpression of HLX: analyses of sprout lengths were performed by measuring the total lengths of sprouts for 10 spheroids each on microscopic images using the ImageJ software (http://www.uhnres.utoronto.ca/facilities/wcif/imagej/). Results are displayed as mean values ± SEM of the fold induction of sprout lengths observed compared with noninduced samples infected with control viruses (arbitrarily set to 1). One representative experiment of 4 performed is shown. (C) Quantification of inhibition of sprouting by overexpression of UNC5B: Analyses were as described in panel B. One representative experiment of 3 is shown. (D) Quantification of increase in sprouting by down-modulation of UNC5B in endothelial cells infected with Ad.con or Ad.HLX: HUVECs were first transduced with lentiviruses for control shRNA or shRNA targeted against UNC5B. After 1 day, cells were also infected with 15 MOI of Ad.con or Ad.HLX. The following day, spheroids were prepared and the next day incorporated into collagen gels in the presence of VEGF-A. Total sprout lengths of 15 spheroids each were measured. The shown values are calculated from 4 independent experiments and display the relative sprout length ± SEM in comparison to the samples transduced with Ad.con and shRNA.con. ***P < .001, t test.