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Fig. 7 Ectopic expression of ZBP-89 impairs angiogenesis in mouse EB cultures. (A) Primary day 11 EBs were dissociated and cultured in collagen matrix with growth factors for endothelial cell differentiation for four days. Histograms (mean±s.d., n=3) show the number of primary day 11 EBs with 0, <5 or >5 branching vessels/EB derived from the D5 and E1 clones, compared with the control. *P<0.01; **P<0.001; NS, not significant. (B) Representative images of control-, E1- or D5-derived sprouting EB colonies. The reduction in the branching vessels (arrows in controls) in EBs derived from overexpressing ZBP-89 clones E1 and D5 is also clearly reflected by the compactness of the respective colonies; fewer branches are produced compared with the controls. (C,D) Flow cytometric analysis of a single-cell suspension of EBs overexpressing ZBP-89 and stained with PE-labeled anti-CD31 (day 6 culture) or anti-VE-Cadherin (day 7 and day 10 cultures) antibodies. ZBP-89 overexpression significantly reduced the number of CD31+ and VE-Cadherin+ angioblasts (similar results were obtained in two other independent experiments).