Cell migration and invasion assays

Cell migration capacity was measured by a cell migration (wound-healing) assay, as described previously (Bao et al. 2012). Cells were seeded in a six-well plate and incubated at 37 °C until they reached 90% confluence. The confluent cells were then scratched with a 200 mL pipette tip and washed with PBS, followed by incubation with Mag in complete medium. After 24 h of incubation, the cells were fixed and stained with 2% ethanol containing 0.2% crystal violet powder (15 min), and randomly chosen fields were photographed under a light microscope. The number of cells that migrated into the scratched area was calculated. Cell invasive potential was studied by calculating the number of cells that invaded through Matrigel-coated transwell polycarbonate membrane inserts as described previously (Verma et al. 2006). In brief, transwell inserts with a pore size of 12 Am were coated with 0.78 mg/mL Matrigel in serum-free medium. Cells were recovered by trypsinization, washed, and resuspended in a serum-free medium. Then, 0.5 mL of the cell suspension (0.5 × 10⁶ cells) was added to duplicate wells. After incubation for 48 h, the cells that passed through the filter were stained using the Hema-3 stain kit (Fisher Scientific, Houston, TX, USA). The cells in 10 random fields were counted under a microscope. Both assays were performed in four biological replicates.