Cell Invasion Assay

Cell invasion assay was performed by a 24-well Transwell chamber with a pore size of 8 μm (Sigma, San Francisco, USA). Cell starvation was incubated for 8 hours before the experiment began. Each chamber was pretreated with 70 μL Matrigel (BD Biosciences, NJ, USA) diluted by the serum-free DMEM medium at the ratio of 1:10. The cells fixed with medium which containing 0.2% FBS were trypsinized after transfection for 24 hours and transferred to the upper Matrigel chamber in 100 μL of serum-free medium supplementing 5×10⁵ cells and incubated for 48 hours, the bottom chamber was filled with medium containing 30% FBS. The chambers were incubated for 48 h at 37 °C in 5% CO₂ atmosphere. The non-migrated cells were removed by cotton swab, then fixed with 95% ethanol for 30 min and stained with 0.1% crystal violet at 37 °C for 25 min, washed twice with PBS. The cells migrated through the chamber were observed by microscope (×40 magnification and ×100 magnification).