Cell migration and invasion assay

For the invasion assay, a Transwell chamber (EMD Millipore) was used to structure the cell invasion model. In summary, 100 µl of Matrigel (BD Biosciences, San Jose, CA, USA), melted overnight and diluted with serum-free RPMI-1640 medium in a 1:6 ratio, was added to the upper chamber of 24-well plates, following incubation for 4–6 h at 37°C with 5% CO₂. The medium was removed, 500 µl of serum-free medium was added to the lower chamber for 30 min at 37°C to hydrate the basement membrane. A549 cells (1×10⁵) treated with ISL (20 µM) or 0.1% DMSO for 48 h at 37°C were seeded in the upper chamber in 100 µl of serum-free medium. The lower chamber was filled with 500 µl of medium supplemented with 10% FBS. Following a 24 h incubation at 37°C, non-invasive cells on the upper surface of the Matrigel were gently scrubbed with a cotton swab and migrated cells on the lower surface were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, stained with 0.1% crystal violet for 20 min at room temperature and recorded for images under a light microscope (Olympus Corporation, Tokyo, Japan; magnification, magnification, ×100). For the migration assay, the same method as the invasion assay was used, only without Matrigel. Each assay was performed in triplicate.