Transwell migration assays were performed using Transwell chamber inserts with a porous polycarbonate membrane (8-μm pore size) (Corning Costar Incorporated, NY, USA). Briefly, the lower side of the filter was coated with fibronectin (10 μg/ml) and blocked with 1% BSA. For invasion assay, the upper side of the filter is coated with Matrigel according to the manufacturer’s protocol. About 5 × 104 cells were added in the upper chamber and allowed to migrate through the filter toward the lower chamber containing the indicated factors. In parallel, the same volume of cell suspension was seeded in cell culture multiwell dishes to check for equal cell loading. After 12 or 24 hours, nonmigrated cells on the upper side of the filter were removed by a cotton swab, followed by fixing with 11% glutaraldehyde and staining with crystal violet. Microscopic images were then quantified either by cell counting or by converting to a binary image and quantifying the integrated pixel values using ImageJ [National Institutes of Health (NIH)]. Experiments were repeated at least three times in replicates, showing consistent results.

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