Invasion assay

In vitro cell invasion assay was performed using an 8-μm BD Falcon^TM cell culture insert (BD Biosciences) [68]. Aliquots of 1 × 10⁵ cells were seeded into the upper compartment of the chamber. The lower compartment was filled with DMEM medium supplemented with 10% FCS. After 24 h of incubation, the noninvasive cells were removed from the upper surface of the membrane, while the invasive cells were stained with 0.1% crystal violet. Cell images were captured using an Olympus IX71 inverted microscope (Olympus Corp., Tokyo, Japan), under which the cells were counted at 100-fold magnification.