Effect of 7-Cyclo in zebrafish embryos and molecular targets. (A) Effect of 7-Cyclo on zebrafish embryos. Tg(fli1a:EGFP)y1 Danio rerio were incubated for 8 h at the concentrations shown (A1–A4) and starting at 22–23 hpf. Insets show the effect of 7-Cyclo in ISVs reaching the DLAV (arrowheads). (Scale bars: 100 μm.) (B) Embryos were scored for the number of ISVs along the anterior–posterior axis, the number of ISV’s that reach the DLAV, and for the presence or absence of sprouts at the caudal plexus. Ten or more embryos were tested per experimental group per independent experiment (total of three independent experiments). The data shown are representative of one of three independent experiments. Statistical analyses were performed by one-way ANOVA test followed by a Bonferroni multiple comparisons test. (C) Expression of tyrosine kinases by qRT-PCR. Gene expression was normalized by the expression of GAPDH. Results are mean ± SEM (n = 4). Statistical analyses were performed by a Mann–Whitney test. (D) Microarray analysis showing the expression of tyrosine kinases in hESC-derived ECs, HUAECs, and HAECs. The list of genes is linked to the heatmap. Some of the tyrosine kinases are more highly expressed in hESC-derived ECs than in HUAECs or HAECs (displayed in the zoom of the microarray). (E) Kinase activity on hESC-derived ECs and HUAECs after incubation with variable concentrations of 7-Cyclo. Luminescence is inversely related to kinase activity. Results are mean ± SEM (n = 6). Statistical analyses were performed by one-way ANOVA test followed by a Newman–Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Effect of 7-Cyclo in the vasculature of adult zebrafish. Tg(fli1a:EGFP) Danio rerio caudal fins were resected (dotted line) and analysed 5 days after amputation, in the presence of 0.1 μM 7-Cyclo inhibitor (a.1 and a.4) and in DMSO (a.2 and a.3). The effect of 7- Cyclo is considerably stronger in regenerating vasculature when compared with its effect on preformed vasculature. (b) Fluorescence images were quantified using IMARIS software, surface masks and visual correction to determine vascular area. Results are mean ± SEM (n=6 animals per experimental group). Statistical analyses were performed by one-way ANOVA test followed by a Bonferroni multiple comparisons test. * P <0.01, *** P <0.001. The difference of the differences in "forming vasculature" group or "pre-formed vasculature" are statistical significant (P<0.024), thus, the difference between DMSO and 7-Cyclo for "pre-formed vasculature" group is lower than the difference between DMSO and 7-Cyclo for "forming vasculature" group.