Transwell migration and invasion assay
For evaluation of cell migration, 8-mm pore size culture inserts (Transwell; Costar, High Wycombe, UK) were placed into the wells of 24-well culture plates, separating the upper and the lower chambers. In the upper chamber, 5 × 104 cells suspended in 300 μL of serum-free medium containing different concentrations of icariin were added, and 500 μL of cultured medium containing 10% FBS was added to the lower chamber. For Matrigel invasion assays, 1 × 105 cells were added to the upper chamber pre-coated with Martrigel and containing different concentrations of icariin. After incubation for 24 h at 37ºC, the non-migrated and non-invasive cells located on the upper surface of the filter were removed with a cotton swab, and the migrated and invasive cells on the bottom surface of the membrane were fixed with methanol and stained with 0.1% crystal violet. Finally, the cells were imaged and numbered using a microscope (Olympus, Japan).