Matrigel invasion assay

Cell invasion assay was performed using a 24-well transwell chamber with 8 μm pores (Costar, Cambridge, MA, USA). Inserts were coated with 20 μL of Matrigel (1:3 dilution; BD Biosciences, San Jose, CA, USA). At 48 h after transfection, cells were trypsinized, and 3×10⁵ cells in 100 μL of serum-free medium were transferred to the upper Matrigel chamber for 18 h. Media supplemented with 10% FBS were added to the lower chamber as a chemoattractant. After incubation, cells that passed through the filter were fixed with 4% paraformaldehyde and stained with hematoxylin. Invasive cells were microscopically counted in 10 randomly selected high-power fields.