Transwell Assay

Cell chemotaxis was assessed as described previously (30). 5 × 10⁴ cells of MCF-7, MCF-7/AWP1 KO, MDA-MB231, or T47D cells were loaded into the inner membrane of the upper chamber of a 8-μm pore size Transwell system (Costar, Corning, NY). To evaluate its chemotactic potential, TNF-α (20 ng/ml, R&D systems, Minneapolis, MN) was then added to the lower chamber. After incubation for 24 h to allow for migration, cells that had not migrated were removed from the top of the membrane with a cotton swab. The migrated cancer cells in the outer membrane of the upper chamber were then stained with hematoxylin and counted under a microscope.