Upper chamber

To test cell migration ability, each upper chamber (Biofil, China) was seeded 50 000 cells with serum‐free culture medium. Also, to evaluate cell invasion ability, the upper chamber with 1:7 concentration of Matrigel membrane (BD, USA) was used. The lower chambers were infiltrated in a complete culture medium. Cultured about 72 hours the chambers were collected, using the same way described above to fix and stain the cells, then wiped cells fixed in the upper chamber with a swab.

Cells (8000/well) were seeded into 96-well plates (Servicebio, WuHan, China), and proliferation was evaluated using Cell Counting Kit-8 (Biosharp Biotechnology, Beijing, China) reagent. The optical density was read at 450 nm. For clone formation assays, cells were cultured for 14 days, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 15 min (Meilunbio, Dalian, China). For scratch assays a linear wound was created in a monolayer of serum-starved cells using a 10-μL pipette tip, and cell coverage across this line was determined. For Transwell migration assays, cells were seeded into the upper chamber (8 μm; BIOFIL, Guangzhou, China) and incubated with serum-free medium. The lower chamber contained medium with 10% FBS. After 24 hours, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.