Matrigel coated 24 well transwell plates

Transwell plates (8-µm pore size; Corning, Inc.) were used for the migration assay and Matrigel-coated 24-well Transwell plates (cat.no. 354480; Corning, Inc.) were used for invasion assays. Following 48 h of transfection, Huh-7 cells (2x10⁴) were incubated in serum-free medium for starvation and seeded into the upper chamber of the Transwell chambers, whilst 600 µl DMEM supplemented with 10% FBS was added into the lower chambers. Following incubation at 37˚C in a 5% CO₂ atmosphere for 24 h, the cells that remain in the upper chamber were removed with a cotton swab whereas cells in the lower chamber were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 10 min. The number of migratory and invasive cells were counted in five randomly selected fields of view using an light inverted microscope at χ100 magnification (TS100; Nikon Corporation).

To evaluate cell invasion, 2 × 10⁵ MG-63 cells or 1 × 10⁵ HOS-MNNG cells were placed in serum-free DMEM in the presence or absence of 4 μM thiostrepton in the upper chamber of Matrigel-coated 24-well Transwell plates (Corning Inc., Corning, NY, USA). The lower chamber was filled with 600 μL of DMEM containing 20% FBS. After incubation at 37°C for 24 h, the cells that had migrated to the lower surface of the membrane were fixed in 100% methanol for 30 min, stained with 0.1% crystal violet for 20 min, and counted under a microscope. Migration assays were carried out in uncoated 24-well Transwell plates (Corning), following the otherwise identical experimental procedure. The invasion and migration capacities of MG-63 or HOS-MNNG cells stably expressing a FoxM1-targeting shRNA or NTC were assessed using the same methods in the absence of thiostrepton.