Transwell migration and invasion assay

The transwell insert with millipore membrane (pore size of 8 μm, Falcon, BD Franklin Lakes, NJ, USA) was used. For transwell migration assay, 2 × 10⁵ A549 cells and 5 × 10⁵ CL1–0 cells were seeded onto the upper chamber with 1 ml serum-free medium. For transwell invasion assay, the transwell inserted membranes were pre-coated with Matri-gel (2.5 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) 1 day before seeding cells. Complete medium with 20% FBS was supplemented into the lower chamber as chemoattractants. The cells were incubated for 16 ~ 24 h and then the cells attached on the reverse side of the membrane were then fixed and stained. Six random views were photographed and quantified under an upright microscope (Nikon E400, Yurakucho, Tokyo, Japan).