Transwell invasion and migration assay

To further examined the cell invasion and migration, the transwell invasion and migration was performed using 8 μm pore size transwell chambers (Corning, USA) in vitro following the manufacturer's instructions. In brief, the matrigel (5 mg/ml, Corning, USA) was diluted into 1 mg/ml in ice-cold RPMI 1640 medium supplemented with 10% FBS. An aliquot of 200 μL diluted matrigel was added to the upper transwell chambers and incubated at 37°C for 4 h for gelling. A total of 1 × 10⁵ cells in 400 μL media supplemented with no FBS were plated in the upper chamber and 600 μL RPMI 1640 medium supplemented with 10% FBS was covered on the bottom chambers as chemo attractant. After incubation at 37°C for 48 h, the non-invasive cells in the top surface were carefully removed with a cotton swab. The invasive cells that had traversed to the bottom surface were fixed in dehydrated alcohol for 30 min and stained with 4 mg/ml crystal violet for 10 min. To quantify the traversed cells, cell counting was obtained by photographing 5 random fields under microscope at 400× magnification [27]. The migration assay was performed in a similar strategy with chamber membrane without coating with matrigel.