Quantitative analysis of endothelial sprouting in response to angiogenesis inhibitors. For inhibition studies of EB endothelial sprouting, VEGF (50 ng/ml) and FGF2 (100 ng/ml) were added at day 0 and at day 6 as indicated in the Methods. At day 6, EBs were also treated by the indicated angiogenesis inhibitors or by their respective vehicle (VHL). Analysis of the mean total sprout length of angiogenic EBs was performed at day 11 on 39, 25, 26, 18, 55 and 76 EBs treated with VHL TGFβ, 10 ng/ml TGFβ, VHL PF4, 2.5 μg/ml PF4, VHL angiostatin and 2.5 μg/ml angiostatin, respectively. In each condition, data represent the mean values ± SE from one out of two differentiation experiments performed in collagen gel with CJ7 ES cells. * p < 0.05, *** p < 0.001; significantly different from respective control values by unpaired Student's t test.