Vegf a165

The inhibitory effect of compound on the viability of HUVECs was measured using a cell-counting assay (cell counting kit (CCK)-8, Dojindo, Kumamoto, Japan) according to the method of Huang et al. [28 (link)]. Briefly, HUVECs (6000 cells per well) were seeded in a 96-well plate for overnight attachment. The growth medium was replaced with fresh medium containing various concentrations of compounds. After incubation for 48 h, the medium was replaced with DMEM/F12 containing 10% 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8). After being further incubated for 4 h, the absorbance was measured at 450 nm with a microplate reader (Thermo Fisher Scientific, New York, NY, USA).
The inhibitory effect of compounds on the viability of VEGF-induced cells viability was also measured using the cell-counting assay. HUVECs (6000 cells per well) were seeded in a 96-well plate. After incubation with 50 ng/mL of VEGF (VEGF-A165, PeproTech, Jersey City NJ, USA) in the presence or absence of various concentrations of compounds for 48 h, the medium was replaced with DMEM/F12 containing 10% WST-8. After being further incubated for 4 h, the absorbance was measured at 450 nm with a microplate reader. The cells incubated only with medium were set as a vehicle control.

All GFs and chemokines were purchased in their mature forms, highly pure (>95% pure), carrier-free, and lyophilized^{35 (link)}. VEGF-A121, VEGF-A165, PlGF-1, PlGF-2, PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, FGF-1, FGF-2, FGF-6, FGF-7, FGF-9, FGF-10, FGF-18, BMP-2, BMP-3, BMP-4, BMP-7, β-NGF, NT-3, BDNF, IGF-1, IGF-2, HB-EGF, CXCL-11, and CXCL-12α were purchased from PeproTech. CXCL-12γ was purchased from R&D systems. Except for PDGF-DD and BMP-7, which were produced in eukaryotic cells, all GFs were produced in Escherichia coli and thus were not glycosylated. All GFs were reconstituted and stored according to the provider’s instructions to regain full activity and prevent loss of protein.

HBVPs were seeded in 96-well plates at a density of 1 × 103 cells/well (100 μL/well). After allowing cells to attech for 24 h, HBVPs were treated with lenvatinib at different concentrations, 20 ng/ml PDGF-BB (PeproTech Inc.) and 60 ng/ml VEGFA165 (PeproTech Inc.). At the testing time points (24, 48 and 72 h), 10 μL of CCK-8 solution (Dojindo Laboratories) was added to each experimental well and then incubated for 2 h at 37°C. The absorbance was measured at 450 nm with an automatic microplate reader (Molecular Devices LLC, San Jose, CA, USA).

HUVECs viability was determined using the cell counting kit-8 (CCK-8) cell viability assay (Dojindo Laboratories, Kumamoto, Japan). HUVECs (3×103 cells per well) were seeded in flat-bottomed 96-well plates. After incubation for 24 h, the cells were treated with lenvatinib at various concentrations and 60 ng/ml VEGFA165 (PeproTech Inc.) in serum-reduced (1%) RPMI 1640 medium for 72 h. The final concentration of dimethyl sulfoxide (DMSO, as vehicle) was ≤1%. Results were expressed as the mean ± S.E.M. of at least three independent proliferation assays. The dose-response curve and half maximal inhibitory concentration (IC50) values of lenvatinib were calculated busing the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).