Transwell assay

Cell migration was performed in Transwell chambers (Corning Inc., Corning, USA) as described in previous studies [14, 45]. Briefly, cells in serum-free DMEM were placed into the upper chambers of Transwell inserts set within wells with 8 μm pore filters, and incubated at 37°C for 12 h. The migrated cells on the lower membrane surface were fixed in methanol and stained with 0.1% Giemsa stain. Eight microscope fields from each Transwell chamber were randomly selected, and cells adhering to the undersurface of the filter were imaged and counted using an Olympus BX41 microscope equipped with a digital camera (Olympus, Tokyo, Japan).