Fibronectin coated filter

For the migration assay, infected cells were seeded into the upper chamber of a Transwell with a fibronectin-coated filter (8-mm pore size, Corning Life Sciences). The bottom chamber contained medium containing 10% FBS. After a 20-hour incubation, cells adherent to the upper surface of the filter were removed using a cotton swab, and those attached to the bottom of the membranes were stained with crystal violet following fixation with methanol.

For the transwell assay, 24 h after transfection, A549 or H1299 cells were serum-starved for 6 h. Next, 3 × 10⁴ cells (for A549 cells) or 5 × 10⁴ cells (for H1299 cells) in 200 μL serum-free medium were seeded into the upper chamber of a transwell plate with a fibronectin-coated filter (8 mm pore size; Corning Life Sciences, Corning, NY, USA). The lower chamber contained medium supplemented with 10% FBS. After 24 h of incubation at 37 °C, non-migrated cells were scraped off of the filter using a damp cotton swab. Following fixation with 4% paraformaldehyde, migrated cells were stained with crystal violet. The number of cells in six randomly chosen fields was counted. Each assay was performed in triplicate wells and repeated three times.
For the wound healing assay, H1299 and A549 cells were infected with NC/Plasmid-FUZ or transfected with si-NC/si-FUZ for 48 h and then transferred into serum-free medium. When the cell fusion reached 90%, the middle of each well was scratched with a standard 10 μL pipette tip. At each indicated time point, photographs of wound closure were taken using an inverted microscope (100× magnification; Nikon, Japan) and subsequently analyzed using ImageJ software V1.8.0.112 (National Institutes of Health, Bethesda, MD, USA).

For the migration assay, passage 3 BMSCs were seeded into the upper chamber of a Transwell plate with a fibronectin-coated filter (8-mm pore size, Corning Life Sciences). The bottom chamber contained medium supplemented with 10% FBS. After incubation for 18 h in 37 °C, cells adherent to the upper surface of the filter were removed. The cells attached to the bottom of the membranes were fixed with methanol and stained with crystal violet.