Matrigel-based Invasion Assay for Cell Migration

The Matrigel-based invasion assay was performed as previously described [33 (link)]. Briefly, 2×10⁴ cells were plated in the upper Transwell chamber (Corning, NY, USA) in DMEM containing 1% FBS. The chamber basement membranewas coated with 60 μg Matrigel (Matrigel, Becton-DickinsonBiosciences, NJ). Then, the chamber was inserted into 24-well plates filled with 600 μL of DMEM medium containing 10% FBS. After incubation at 37°C for 24 h, the cells on the upper portion of the chamber membranes were removed, and the lower surfaces of the chamber membranes were fixed with 5% glutaraldehyde for 5 min and stained with 5% Giemsa. The number of invading cells from four randomly selected fields was counted under a microscope. For rescue assays, the A549 and H322 cells with stable expression of KLF17 protein were again transfected with pReceiver-M15-uPA containing uPA cDNA or a control vector; For effects of Src and p38/MAPK inhibition on invasion assay, the upper Transwell chamber (Corning, NY, USA) in DMEM containing 1% FBS and different concentration of SB 203580 (p38/MAPK inhibitor) or HY-13805 (PP2, Src inhibitor), and then the invasiveness of the cells was analyzed by Transwell assays in the same manner.