8 μm polyethylene terephthalate membrane filters

Cell migration and invasion assays were performed in a 24-well transwell plate with 8-μm polyethylene terephthalate membrane filters (Costar, Corning, MA, USA). GBC-SD (2×10⁴) and NOZ (3×10⁴) cells in 500 μL of serum-free medium were added to the upper chambers, which contained either uncoated or Matrigel-coated membranes. Each lower chamber was filled with 500 μL medium with 10% FBS. After 24 h of incubation, the filters were removed, and the non-migrating cells on the upper sides of the filters were detached using cotton swabs. The filters were fixed with 4% paraformaldehyde for 15 min. The cells located in the lower filters were stained with Giemsa for 20 min. Migrated or invaded cells were counted in five randomly chosen fields (×100 magnification) per well. For wound healing assay, transfected GBC-SD and NOZ cells were seeded into 6-well plates and grown to confluence. Wounds were created by scraping the confluent cell monolayers with a 200-μL pipette tip. After the cells were extensively rinsed to remove cellular debris, they were cultured in FBS-free media. Photomicrographs were taken under a microscope (Leica) at 0 and 48 h. The percent change in migration was determined by comparing differences in wound width.

A Transwell apparatus was used with 8-μm polyethylene terephthalate membrane filters (Corning Inc., USA). The upper chambers were seeded with 200 µl of serum-free medium containing serum-starved cells (HepG2 and SK-Hep-1: 1 × 10⁴ cells; Huh7: 2 × 10⁴ cells). The lower chambers were filled with 500 µl of 10% FBS-DMEM. After 24 hours, cells that invaded the lower chamber were fixed and stained with 0.2% crystal violet (Beyotime Biotech) as previously described (36 (link)). The invaded cell number from experiments in triplicate was counted in five randomly selected fields per chamber under an inverted microscope (Leica, Germany).