In vitro cell proliferation assay

Using the transfected A375 cells, cell proliferation was analyzed using a water-soluble tetrazolium 8 (WST-8)-based colorimetric proliferation assay kit (Cell Counting Reagent SF; Nacalai Tesque). The cells were seeded in triplicate at a density of 5,000 cells in 200 μl of culture medium supplemented with 5% FBS in 96-well plates, incubated for 24, 48, 72, or 96 hours, and cell viability was assessed in accordance with the manufacturer's protocol. Briefly, cells were washed gently with PBS three times and non-adherent or dead floating cells were removed. The cell count reagent was added to each well and the plates were incubated at 37°C for 3 hours to allow the conversion of the reagent to formazan by mitochondrial dehydrogenase. Formazan was quantified by measuring the absorbance at 450 nm using a microplate reader (FlexStation 3; Molecular Devices, Tokyo, Japan).