Cell Motility and Invasion Assay

Cell motility and invasion abilities were examined using Boyden Chamber Assay (Corning, United States). Briefly, for cell motility assay, 200 μl HCC cells (1 × 10⁶/ml) pretreated with metformin were suspended in a serum-free medium and delivered into the upper chamber. A complete culture medium with 20% FBS was added into the lower chamber as a chemoattractant. After 36 hours of culture, the migrated cells at the bottom membrane were fixed by 4% paraformaldehyde and stained with crystal violet. The number of migrated cells in five random fields was counted under a light microscope. For cell invasion assay, the procedures were the same as that of migration assay except for the Matrigel-coated bottom membrane (Corning, NY, United States) of the upper chamber and 48 h cell culture.