Matrigel

200 μl Matrigel (Corning, USA) was plated in the wells of 24-well plate, then the plate was placed at 4 °C overnight to make the Matrigel evenly distributed. 2 × 10⁴ endothelial cells was seeded on the Matrigel. Following a 4-h culture, the medium was replaced with fresh high-glucose DMEM to eliminate any unattached cellular debris. Subsequently, 10 μl of Calcein AM fluorescent dye (KeyGen, China) was added 10 min before fluorescence microscopy observation to improve the visibility of cell frame on Matrigel. Subsequently, images were captured using a fluorescence microscope. The angiogenesis ability of endothelial cells was quantified by counting branching points in the randomly selected fields from each group using ImageJ software.

Cells in culture dish were preincubated with Mitomycin-C (10 μg/ml) for 1 h at 37 °C to suppress proliferation. For the transwell migration assay, 2 × 10⁴ cells in 100 μl serum-free medium were seeded into the upper chamber of 8-μm transwell inserts (BD Biosciences, Franklin Lakes, NJ). In the lower chamber 500 μl medium containing 10 % bovine serum albumin was added. After 20 h of incubation at 37 °C, cells in the upper chamber were removed carefully. While cells adhering to the underside of the membrane were fixed in methanol for 15 min and then stained with 0.1 % crystal violet (KeyGEN biotech) for 30 min. For the transwell invasion assays, 2 × 10⁵ cells in 100 μl serum-free medium were put into the upper chamber, which pre-coated with 50 μl Matrigel (BD Biosciences, Bedford, MD) diluted 1:4 with serum-free medium. While medium containing 10 % bovine serum albumin were in the lower chamber. After 24 h of incubation at 37 °C, Matrigel and cells in the upper chamber were removed carefully, while cells adhering to the underside of the membrane were fixed in methanol for 15 min and then stained with 0.1 % crystal violet (KeyGEN biotech) for 30 min.
The number of cells was counted in 5 randomly selected visual fields under an inverted microscope DMI4000B (Leica, Wetzlar, Germany).

An 8μm pore size transwell chamber mixed with Matrigel (Keygen, Nanjing, China) was used for transwell chamber assay. Cells were digested and counted. A total of 1 × 10⁶ cells in 100 μL medium supplemented without FBS were plated in the upper chamber and 500 μL medium supplemented with 10% FBS was covered on the bottom chambers as chemo attractant. After 24 h incubation in a humidified incubator, non-migratory cells on the upper membrane surface were carefully removed, and those on the bottom surface were fixed with 4% polyoxymethylene (Sigma, MO, United States) and stained with 0.1% crystal violet (Sigma, MO, United States) for 15 min. Cells were counted by photographing 5 random fields under a microscope (BX53, Olympus, Tokyo, Japan) at 400 × magnification and images were record.