BMX inhibited VEGF-induced cell proliferation in HUVECs.

<p>(A) HUVECs were starved in 2% FBS containing medium without ECGS for 16 h. After starvation, cells were pretreated with indicated concentrations of BMX followed by the stimulation with VEGF (20 ng/ml) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± S.E.M. of five independent experiments performed in duplicate *<i>p</i><0.05, compared with the group treated with VEGF alone. (B) HUVECs were treated as in (A), and cell proliferation was determined as described in the “<i>Materials and Methods</i>” section. Each column represents the mean ± SEM of five independent experiments performed in duplicate. *<i>p</i><0.05, compared with the group treated with VEGF alone. (C) HUVECs were treated as in (A), and cytotoxicity of BMX was determined by LDH assay. Cells were also treated with cell lysis buffer (total lysis) to serve as positive control. (D) HUVECs were treated as in (A), the percentage of apoptotic cells was then analyzed by flow-cytometric analysis as described in the “<i>Materials and Methods</i>” section. The lower left quadrant of each panel (annexin V⁻PI⁻) shows the viable cells, which exclude PI and are negative for annexin V binding. The lower right quadrant (annexin V⁺PI⁻) represents the early apoptotic cells, annexin V positive and PI negative, demonstrating cytoplasmic membrane integrity. The upper right quadrant (annexin V⁺PI⁺) contains advanced apoptotic cells and necrotic cells, which are positive for annexin V binding and for PI uptake. Results shown are representative of four independent experiments.