Plgf 2

HUVEC and hepatoma cells (HuH-7, KYN-2, HAK1-B, Hepa1-6) were seeded (1 × 10³/well) in 96-well plate containing a suitable medium [EGM-2 supplemented with 5% fetal bovine serum (FBS) for HUVEC and Dulbecco’s modified Eagle’s medium with 10% FBS for hepatoma cells]. After 24-hour incubation at 37°C, the media were replaced with media containing various concentrations of recombinant VEGF-165 (0, 0.1, 1, 10, 100 pM, 1 nM) (PeproTech, Inc., Rocky Hill, NJ) or PlGF-2 (0, 0.1, 1, 10, 100 pM, 1 nM) (PeproTech, Inc.) and 5% FBS for HUVEC cell proliferation assay. To inhibit HUVEC and hepatoma cell proliferation, the media were replaced with media containing various concentrations of aflibercept (0, 10, 100 pM, 1, 10, 100 nM) combined with recombinant VEGF-165 (0.2 nM) or PlGF-2 (1.0 nM), and 5% FBS. After 72-hour incubation, cell proliferation was evaluated by the tetrazolium-based assay (Cell Count Reagent SF; Nakalai Tesque Inc., Kyoto, Japan). The experiment has been done at least twice to confirm reproducibility.