Cell Culture Optimization and Gene Expression Analysis

B16-F10 and HaCaT cells (5 × 10⁴ cells/well) were plated on 12-well plates and incubated. Then, B16-F10 cells were treated with 1% (v/v) CFS or arbutin (200 µM) for 6 h and 40 h in the presence or absence of 200 nM α-MSH. HaCaT cells were pretreated with CFS (1 and 3% (v/v)) for 30 min and then co-treated 100 µM H₂O₂ for 24 h. Then, the cells were harvested and washed twice with phosphate-buffered saline. Total cellular RNA was prepared using TRIzol solution according to the manufacturer’s instructions. RNA was converted to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The 2X GreenStar qPCR Master Mix (Bioneer, Daejeon, Korea) was used in all the samples, and reactions were carried out in a 20 µL final reaction volume. Each experiment was performed at least twice in duplicates using the following primers in Supplementary Table S1. All gene expression levels were calculated by using the Ct value by the method 2^−ΔΔCt (where ΔCt = Ct_{[target gene]} − Ct_[GAPDH]).