Fig. 3

The antagonistic effect of Y332D on the activation of VEGF/VEGFR pathway, VEGFA-induced proliferation and tube formation in HUVECs. a NFAT-luciferase reporter assay was performed to show the blockade effect of Y332D on VEGF/VEGFR pathway. HEK-293 cells overexpressing VEGFR2 were transfected with the lentiviral vectors carrying the NFAT and luciferase gene (NFAT-RE-Luci) to construct stable transfected cell lines 293-NFAT. 293-NFAT cells were cultured in 2% FBS-DMEM with VEGFA (20 ng/ml) and serially diluted Y332D or controls for 6 h. Then, the luminescence was detected. b Luminescent cell viability assay was performed to measure the inhibitory effect of Y332D on VEGFA-induced HUVEC proliferation. 5 × 10³ HUVECs were seeded in 96-well plates overnight at 37 °C. Then, the medium was replaced with endothelial cell basal medium mixed with VEGFA (50 ng/ml) and serially diluted Y332D or controls. Cell viability was detected after incubation at 37 °C for 72 h. c Tube formation assay was performed to show the inhibitory effect of Y332D on VEGFA-induced vessel-like tube formation. 2 × 10⁴ HUVECs were seeded in 96 well flat-bottom plates after plates were precoated with 50 μl Matrigel for 30 min at 37 °C. The cells were incubated in endothelial cell complete medium mixing with 100 ng/ml VEGFA and 10⁶ pM antibodies or control for 12 h at 37 °C. Then, HUVECs were fixed with 4% paraformaldehyde for 15 min. The images of tube-like structures were captured with inverted microscope. Bars, SDs; α-TGF-β: anti-TGF-β, α-VEGF: anti-VEGF