Transwell migration and invasion assays

For migration assay, transfected cells (40,000/well) were placed into the upper chamber of transwell insert with an 8 μm pore size (MilliporeSigma, Canada) with 200 μl of serum-free DMEM medium, 800 μl of DMEM with 10% FBS was added to the lower chamber. After culture for 24 hours, cells that migrated to the lower membrane surface were fixed in 4% paraformaldehyde for 30 min, and then stained with 1% crystal violet for 15 min. A light microscope (Olympus, Tokyo, Japan) was used to photograph and calculated migrated cells.

For the invasion assay, transfected cells (40,000/well) with 200 μl of serum-free DMEM medium were plated into the upper chamber coated by Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), 800 μl of DMEM with 10% FBS was added to the lower chamber. After culture for 72 hours, the cells that invaded the lower membrane surface were removed and analyzed in the migration assay. The experiments were performed in triplicate.