Transwell invasion and migration assays

Transwell chambers (pore size, 8 mm; EMD Millipore) were used to detect the invasion and migration of the ES cells transfected with siNKAP or pcDNA-NKAP. For cell invasion, assay the chambers were pre-coated with Matrigel (BD Biosciences) at 4°C for 30 min. Following transfection, A673 or RD-ES cells were seeded in the upper chambers at a density of 1×10⁵ in 200 µl of serum-free DMEM. The lower chambers were then filled with 500 µl of DMEM with 20% FBS as the chemoattractant. Following 24 h of incubation at 37°C, the non-invaded cells on the upper Transwell membrane were removed by scraping, while the invaded cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 20 min. The stained cells were photographed using a light microscope at magnification ×100 and counted in three random view fields.

The cell migration experiment followed the aforementioned protocol however no Matrigel was used in the Transwell chambers.