Scratch wound-healing and Transwell assays

Cell migration and invasion abilities were assessed by scratch wound-healing and Transwell assays, which were performed as previously described [28, 29]. Briefly, for the scratch wound-healing assay, different lentivirus-infected GBC cells were seeded in 96-well plates (approximately 5 × 10⁵ cells/well). The next day, after the cells had adhered to the bottom, linear wounds were created by scratching the centre of the cell monolayer. At different time points, wounds images were obtained using a Celigo instrument, and the migration area of different lentivirus-infected GBC cells was analysed by this software. Briefly, for the Transwell assay, different lentivirus-infected GBC cells in 0.2 mL serum-free medium (approximately 1 × 10⁵ cells/well) were added to the upper chamber of 24-well plates containing 8-μm pores (Corning, 3422), and 0.6 mL DMEM (Corning, 10–013-CVR) containing 30% FBS (Ausbian, A11–102) was added to the lower chamber. The plate was incubated for 24 h after which non-invading cells were removed with cotton swabs; the remaining cells were fixed in 4% paraformaldehyde for 30 min, stained with 0.5% crystal violet, and counted under a microscope at 200× magnification.