Cell migration and invasion assays

Wound healing and Transwell assays (without Matrigel coating) were performed to evaluate cell migration. The Transwell assay [with Matrigel coating (BD Biosciences)] was performed to assess cell invasion. For the wound healing assay, transfected cells were cultured to 90–95% confluency in 6-well plates and scratched using 200-µl pipette tips. Migration images were captured at 0 and 36 h after scraping with an inverted microscope (magnification ×200; Olympus Corporation) and analyzed objectively via Image J v1.46 software (National Institutes of Health). For the Transwell assay, 5×10⁴ transfected cells in FBS-free RPMI-1640 medium were seeded into the top chamber, while the lower chamber was supplemented with RPMI-1640 medium containing 20% FBS. After culturing for 36 h, the non-migrated or non-invasive cells were removed with a cotton tip, and the remaining cells on the bottom surface were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet for 15 min at room temperature. The stained cells were imaged in five randomly selected fields under an optical microscope (magnification ×400; Olympus Corporation) and counted using ImageJ v1.46 software (National Institutes of Health).