Figure 3

In vitrobiological activity. (A) HB-002.1 inhibited VEGF-induced VEGFR2 phosphorylation as revealed with immunoblotting assay. This experiment was repeated three times, all showing a similar pattern of inhibition in VEGFR2 phosphorylation. (B) Inhibition of VEGF-induced HUVEC cell proliferation was analyzed with the CCK-8 kit, a colormetric assay. Assay was repeated three times with duplicate wells for each concentration. Representative assay is shown. Significant differences between HB-002.1 and hIgG-Fc (P < 0.05 at all except lowest dose), and bevacizumab and hIgG-Fc (P < 0.05 at all doses) were observed. Bevacizumab and hIgG-Fc was used as positive and negative controls, respectively. (C) Representative microscopic images of HUVEC tube formation in Matrigel are shown for medium alone, or for medium plus VEGF with or without HB-002.1, Bevacizumab, or hIgG. (D) Tube formation was quantified by counting the total vessel length per field. Data were collected from duplicate wells (mean ± standard deviation). Statistical significance was evaluated by ANOVA and Tukey’s multiple comparison test. Differences between Medium versus VEGF and VEGF + HB-002.1 versus VEGF + Bevacizumab were not significant (NS). Differences between VEGF + HB-002.1 or VEGF + Bevacizumab versus Medium or VEGF + hIgG were significantly different (P < 0.05).