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FIGURE 5 NB-5-MT (2) suppresses angiogenesis in vivo. (A-B) Inhibitory effect of 2 on retinal vascularization in DMOG-treated Tg(fli1a:EGFP) zebrafish larvae. A, Representative images of retinal vascularization. DMOG-mediated retinal vascularization was decreased by treatment with 1 mM of 2. Nuclei were stained with 1 μg/ml DAPI (PBS). Scale bar, 100 μm. B, Quantification of vessel diameters in zebrafish retinas. Data are expressed as the mean ± SD (n = 20). **P <.01, vehicle vs. DMGF; +P <.05 and ++P <.01, DMOG vs. DMOG plus compound. C, Inhibitory effect of 2 on VEGF and HIF-1α mRNA expression in DMOG-treated zebrafish larvae. D, Inhibitory effect of 2 on HIF-1α protein expression in zebrafish larvae. (E) Inhibitory effect of 2 in an in vivo Matrigel plug assay. Matrigel plugs were prepared with bFGF alone, bFGF plus melatonin, or bFGF plus 2, and implanted into C57/BL6 mice. The mice were euthanized 15 days after implantation, and the plugs were recovered. Representative images of macroscopic Matrigel plugs (top panels) from 3 independent experiments. Scale bar, 0.5 mm. Hemoglobin levels in the Matrigel plugs (bottom) were determined using Drabkin's solution. The hemoglobin content was normalized to the weight of the plug. Data are expressed as the mean ± SD (n = 6). *P <.05, vehicle vs. bFGF; +P <.05, bFGF vs. bFGF plus compound