Wound healing assay

Cells were cultured to 6-well plates (2 × 10⁵ cells/well) until about 100% confluence. 100 nM apatinib or 100 ng/ml rhVEGF + 100 nM apatinib were added into medium and cultured 24 h. 200 μl pipette tip was used to create a wound gap on cell monolayer, and Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan) at 100 times magnification was used for imaging immediately. Migration was then observed 24 h post wound scratched. Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculated the relative migration distant% as: [(The relative distance recorded at 0 h - the relative distance recorded at 24 h)/the relative distance recorded at 0 h] × 100.