Control inserts

Transwell invasion/migration assay was conducted according to the manufacturer’s protocol using BioCoat Matrigel Invasion Chamber, Cell Culture Inserts, and Control Inserts (Corning). Approximately 1.0 × 10⁴ cells were seeded in the insert chamber and incubated at 37 °C for 48 h. The insert chamber membranes were then fixed with ice-cold methanol and stained with hematoxylin and eosin.

Transwell invasion/migration assays were performed using BioCoat Matrigel Invasion Chamber, Cell Culture Inserts, and Control Inserts (Corning) in accordance with the manufacturer's protocols. For invasion/migration assay, 4 × 10⁴ to 10⁵ cells were seeded in the insert chambers and cultured at 37 °C for 24 h. The membrane of the insert chambers was fixed by ice‐cold methanol and stained by hematoxylin and eosin.

The invasion assay was made using the 24-well plate growth factor reduced Corning^® Matrigel^® Invasion chambers 8 μm pore size (Corning, Bedford, MA, USA) and 24-well Control Inserts 8 μm pore size (Corning) according to manufacturer’s instructions. Briefly, cells (1 × 10⁵) in 500 µL serum-free medium were plated into the upper chamber and the bottom wells were filled with 750 µL complete medium. The cells were incubated in 5% CO₂ at 37 °C for 16 h. Then, cells in the upper chamber were removed using cotton swabs and the cells invading the bottom of the membrane were fixed with 4% paraformaldehyde for 20 min. The nuclei were stained with DAPI (Sigma-Aldrich, Darmstadt, Germany) 1 µg/mL plus 1% Triton X-100 in 1X PBS (Gibco, Paisley, UK) for 15 min followed by two washes in Milli-Q H2O. Ten random fields from each membrane were photographed using the Zoe fluorescent cell imager (Bio-Rad) and the cells’ nuclei were counted using the Analyze Particles tool from ImageJ software.