Well transwell plates

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.

Cell invasion assays were performed using Matrigel-coated (invasion assay) 24-well Transwell plates (8.0 μm pore size; Corning, USA). Briefly, MDA-MB-231 cells (2 × 10⁴ cells/well) in the upper chambers were treated with different concentrations of pristimerin (0.1, 0.2, or 0.3 μM) and 700 μL L-15 media containing 5% FBS was added to the bottom chambers. After 24 h of culture, the cells that did not traverse the filter were removed using cotton swabs, and the cells that traversed the filter were fixed using 4% paraformaldehyde and dyed using 1% crystal violet.