Wound-Healing and Transwell Assays

The migration of XGC-1 cells was analyzed by wound-healing and transwell assays. For the wound-healing assay, wounds on cell layer were created using a pipette tip (100 μL) when the cells reached 90% confluence. Then, FBS-free medium was used to rinse the created wound to remove all exfoliated cell debris. Photographs were taken at 0 h and 24 h with an inverted microscope (Nikon). Percentage of migration was calculated according to the following equation: cell migration rate (%) = (1-the distance following healing/the distance prior to healing) × 100%.

For the transwell migration assay, serum-free medium containing transfected XGC-1 cells (1×10⁵ cells) was added to the top chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was added to the bottom chamber. After removing the cells on the upper surface of the membrane, the remaining cells were fixed with paraformaldehyde (4%, Sigma) and stained with crystal violet (0.5%, Sigma). The number of migrating cells was calculated with an inverted microscope (Nikon) at 100 × magnification.

The procedure of the transwell invasion assay was the same as the transwell migration assay. It was worth noting that the transwell chamber used in the invasion assay had been pre-coated with Matrigel (Sigma).