Fig. 1

The effects of BD0801 on VEGF/VEGFR2 binding, downstream signaling, and HUVEC cellular functions in vitro. (A) Blockade of VEGF-VEGFR2 binding by BD0801 or bevacizumab (Avastin) was analyzed by ELISA. Results of three independent experiments. (B) Different concentrations of BD0801 or bevacizumab were incubated with 50 ng/ml VEGF for 2 h at 37 °C before they were added into the HUVEC culture. After 72 h, the inhibition of HUVEC proliferation was detected by CellTiter Glo staining. The experiment was performed three times, and one of the representative experiment results is shown. RLU, Relative light unit. (C) HUVECs were cultured in the inner chamber of the Boyden Chamber Transwell. Different concentrations of BD0801 or bevacizumab were incubated with 20 ng/ml VEGF for 30 min before adding them to the outer chamber. After incubating at 37 °C for 16 h, the migrated cells were stained purple by crystal violet and observed under the microscope. Each condition was performed in triplicates. (D) Quantification of HUVEC migration assay described in (C). (E) Different concentrations of BD0801 or bevacizumab were mixed and incubated with 20 ng/ml VEGF for 30 min before they were added to starved and scratched HUVEC culture. The relative wound densities were monitored and quantified for 24 h. The representative images at 24 h are shown. (F) Different concentrations of BD0801 or bevacizumab were mixed and incubated with 50 ng/ml VEGF for 30 min before they were added to HUVEC culture for 3 min. Protein extracts were separated by Western blot for phosphorylated-ERK1/2 (P-ERK1/2), phosphorylated-VEGFR2 (P-VEGFR2), ERK1/2, and VEGFR2. The three rows above (P-ERK1/2, P-VEGFR2, and β-actin) are from Blot 1; the three rows below (ERK1/2, VEGFR2, and β-actin) are from Blot 2. This Western blot analysis was repeated independently three times, and a representative result is shown. The original uncropped blots for this particular experiment are included in Supplementary S2. (G) The quantification and statistical analysis of the three independent Western blot experiments described in (F) are included. The value for VEGF only group was normalized to one. The quantification of P-ERK1/2, P-VEGFR2, ERK1/2, and VEGFR2 normalized for β-actin respectively are included in Supplementary Figs. S3 and S4. The error bars: standard error of the mean (SEM). *, P < 0.05, **, P < 0.01