Melanin content assay

The effect of 1,5-diCQA on melanogenesis in B16 cells was investigated according to a previously published protocol, with certain modifications (22). More specifically, intracellular melanin content was measured by using the NaOH dissolution method: B16 cells were seeded in a 6-well plate at a density of 2x10⁵ cells/well, and then allowed to attach for 12 h. Then, cells were treated with 1,5-diCQA at 0, 5, 50, 100 µM or 8-MOP at 50 µM for 48 h, and SB203580 (10 µM); PD98059 (1 µM); SP600125 (10 µM) for 2 h prior to 1,5-diCQA application. Cells were washed with PBS and harvested with 100 µl radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) in 1.5 Eppendorf (Ep) tubes. Samples were centrifuged at −4°C and 12,000 × g for 20 min, and the supernatant was obtained to determine the protein concentrations. Sediment was dissolved in 150 µl 1M NaOH (with 10% DMSO) for 1 h at 80°C, and solubilized melanin was measured at 405 nm. The amount of melanin was calculated by normalizing the total melanin values with the protein content.