2.5. Melanin Content Assay

The melanin content was measured as previously described [14], with slight modifications. B16F10 cells (5 × 10⁴ cells) were seeded overnight in a 60-mm dish. Subsequently, the cells were treated with different final concentrations (2.5–10 μg/mL) of PME or IBMX. For inhibition of p38 MAPK signaling, the cells were treated with 10 μM SB 203580 for 1 h prior to 10 μg/mL PME treatment. The cells were cultured for 96 h; after that, the cells were harvested and the cell pellets prepared. The cell pellets were dissolved in 200 μL of 1 N NaOH containing 10% DMSO for 1 h at 80°C. After centrifugation at 12,000g for 10 min at 25°C, the supernatant was collected. The cell lysate (100 μL) was pipetted into a 96-well microplate (SPL Life Sciences, Gyeonggi-do, Korea), and the OD at 490 nm was determined using a VersaMax Microplate Reader. The OD values were normalized to the protein content in the cell lysates. Then the protein concentration was estimated by BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA) in accordance with the manufacturer's protocol.