Cell proliferation assay (BrdU incorporation assay)

HUVECs (2 × 10⁴ per well) were seeded in 96-well tissue culture plates and incubated for 24 h. Cells were then starved in M199 medium containing 2% FBS in the absence of endothelial cell growth supplements for another 16 h. After starvation, cells were pretreated for 30 min with various concentrations of DDA, followed by the stimulation with VEGF-A (25 ng/ml) for another 24 h. Cell proliferation was then determined by bromodeoxyuridine (BrdU) cell proliferation enzyme-linked immunosorbent assay (Millipore, Billerica, MA) based on the colorimetric detection of the incorporation of BrdU, following the manufacturer instructions.