Matrigel coating

Invasion assays were performed in Boyden chamber inserts with Matrigel coating (Becton, Dickinson and Company, USA). Insert membranes were stained with 4′, 6-diamidino-2-phenylindole (DAPI) or a 0.1% crystal violet solution (Sigma, USA). Cells were photographed with a monochromatic Olympus camera. Analysis of intensity of transwell membrane calculated in K counts mm² with Odyssey LI-COR Scanner System (LI-COR Biosciences, USA) was utilized as a measure of invasion.

Cell invasion was measured using 24-well Fluoroblok inserts (8 mm Becton Dickinson). Optimization was performed for SKmel147 and A375 cell lines to identify assay time length and Matrigel concentration. Briefly, siRNA transfected SKmel147 or A375 cells (40,000 cells per insert) were suspended in the serum-free medium over a Matrigel coating (Becton Dickinson), and a medium supplemented with 10% serum was used as a chemo-attractant. Cells that invaded after 6–8 h were stained in 4 μg/ml Calcein AM dye (ThermoFisher) for 1 h and counted in 5 different fields using a fluorescent microscope. For each independent experiment, three replicates per condition were run. The average of cell counts from three replicates per condition was used for plotting results. For control, cells transfected with NTC (siNTC) were present in each experiment. Cell counts for each well were normalized to the mean counts (of replicate wells) for the corresponding condition in the cell input plate to control for cell proliferation effects that may have occurred between initiation of transfection and assay seeding.

Invasion assays were performed in Boyden chamber inserts with Matrigel coating (Becton, Dickinson and Company, USA). Insert membranes were stained with 0.1% crystal violet solution (Sigma, USA) analysed by Cell Sensi Software. Cells were photographed with a monochromatic Olympus camera. Analysis of intensity of transwell membrane calculated in K counts mm² with Odyssey LI-COR Scanner System (LI-COR Biosciences, USA) was utilised after crystal violet staining as a measure of migration and invasion abilities.

For migration and invasion assays, 2 × 10⁴ NPC cells in 500 μl of serum-free DMEM were added to the cell culture inserts using an 8-μm microporous filter with a Matrigel coating (Becton Dickinson Labware, Bedford, MA) for the invasion assay or without Matrigel coating (migration assay). DMEM with 10% FBS was added to the bottom chamber. After 18 h of incubation, the cells on the lower surface of the filter were fixed and stained and then examined on microscopy. Cell counts in five random optical fields (×200 magnification) from triplicate filters were averaged.