Transwell migration and invasion assays

Cell migration and invasion abilities were assessed using transwell chambers with an 8-μm pore size insert (Costar, Cambridge, MA, USA). For transwell migration assay, cells were transfected with 20 nM siRNAs for 48 h and seeded in the upper chambers at a density of 5 × 10⁴ in serum-free medium and the lower chambers were filled with DMEM containing 10% FBS. After incubation for 24 h at 37 °C, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution.

For invasion assay, the upper chambers were coated with 100 μl of 1 mg/ml Matrigel (BD Biosciences). Cells transfected with 20 nM siRNAs for 48 h were seeded on the Matrigel in the upper chambers at a density of 5 × 10⁴ in serum-free medium and the lower chambers were filled with DMEM with 10% FBS. After incubation for 24 h at 37 °C, the invasive cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution.

Images of the stained cells were acquired using a microscope equipped with a CCD camera (Leica). Crystal violet was then extracted with a 10% acetic acid solution, and absorbance was read using SpectraMax 190 Microplate Reader (Molecular Devices) at 570 nm.