Matrigel invasion assay
The invasion abilities of OS cells were evaluated using Transwell invasion chambers precoated with 50 μl of 2 mg/ml Matrigel (BD Biosciences, USA). In brief, 5 × 104 transfected cells suspended in 200 μl of serum-free DMEM were seeded into the upper chambers. A 600 μl volume of DMEM supplemented with 10% FBS was used as the attractant and was added into the lower chambers. After culture for 24 h, cells adhering to the lower surface of the membrane were fixed with paraformaldehyde (4%) and stained using crystal violet (0.1%), whereas cells on the upper surface of the membrane were removed by wiping with cotton swabs. At least three random fields of view containing cells that had migrated or invaded to the lower surface were imaged under an inverted light microscope.