GeGe3 impaired tube formation in vitro and intersegmental angiogenesis of Tg(fli1:EGFP)y1 zebrafish embryos in vivo. (A) Bright-field time-lapse imaging of HUVEC tube formation in the presence of GeGe3. An endothelial tube formation assay was performed in Geltrex™ matrix with VEGF/DMSO or VEGF/GeGe3 for 10 hours. Pictures are shown for the initial cells (0 hours) and after formation of tube-like structures (10 hours). Scale bar = 100μm. (B) Quantification of the effect of GeGe3 on angiogenesis in vitro. The total tube length was determined as well as the network stability index, which is the ratio of total tube length to the number of isolated blocks of segments. Data are presented as mean ± sem. ^*:p-value<0.05. Data are representative of 4 independent experiments. (C) Images of whole 1 day post fertilization (dpf) zebrafish embryos after 8 hours of DMSO or GeGe3 treatment were acquired with a stereomicroscope. Scale bar = 250μm; middle panel: maximal projection of stack fluorescence images of zebrafish tail intersegmental vessels acquired with a confocal microscope, scale bar = 200μm; bottom panel: zoomed fluorescence images of intersegmental blood vessels, scale bar = 100μm. (D) Quantification of GeGe3’s effect on intersegment angiogenesis. The mean length of the intersegmental blood vessels was measured and compared between GeGe3 and DMSO. Data are presented as mean ± sem; ^*:p-value<0.05. Data are representative of three independent experiments with at least five fish embryos per group per experiment.