8 mm polyethylene terephthalate membrane filters

The cell migration assay was performed in a 24-well transwell plate with 8-mm polyethylene terephthalate membrane filters (Corning) separating the lower and upper culture chambers. In brief, melanoma cells were treated as indicated and plated in the upper chamber at 1 × 10³ cells per well in serum-free DMEM medium. The bottom chamber contained DMEM with 10% FBS. Cells were allowed to migrate for 24 h in a humidified chamber at 37 °C with 5% CO2. After the incubation period, the filter was removed and non-migrant cells on the upper side of the filter were detached using a cotton swab. Filters were fixed with 4% formaldehyde for 15 min, and cells located in the lower filter were stained with 0.1% crystal violet for 20 min and photographed.
Cells were counted in four randomly selected fields. Cell counts are expressed as the average number of cells per field of view. Three independent experiments were performed.

In the colony formation test, melanoma cells with overexpression or knockdown of indicated genes were diluted to a single-cell suspension, and 1000 or 2000 cells in each well of six-well plates were incubated with 5% CO₂ at 37 °C for 10 days. The colonies were then stained with 0.04% crystal violet and 2% ethanol and manually quantified.
The melanoma cell migration assay was carried out in a 12-well Transwell plate with 8-mm polyethylene terephthalate membrane filters (Corning). Cells were allowed to migrate in the humidified chamber for 24 h. After the incubation period, the filter was removed, fixed with 4% formaldehyde for 15 min and stained with 0.1% crystal violet for 20 min, and the cells were counted.

Cell migration assays were performed in 24 well transwell plate with 8 mm polyethylene terephthalate membrane filters (Corning) separating the lower and upper culture chambers. In brief, cells were plated in the upper chamber at 5 × 10⁴ cells per well in serum‐free DMEM medium. The bottom chamber contained DMEM medium with 10% FBS. Cells were allowed to migrate for 24 h in a humidified chamber at 37 °C with 5% CO₂. After the incubation period, the filter was removed and nonmigrant cells on the upper side of the filter were detached using a cotton swab. Filters were fixed with 4% formaldehyde for 15 min and cells located in the lower filter were stained with 0.1% crystal violet for 20 min and photographed.