Tri solution

Cell were seeded in 6 well culture plates at a density of 1.5 × 10⁵ cells/well for compound treatment. RNA was isolated using the TRI-Solution™ (Thermo Fisher Scientific), following the manufacturer’s instructions. RT-PCR was performed using the AccuPower® RT PreMix (Bioneer, Daejeon, Korea). 1 µg of mRNA was used for the template. The primer sequences used were as follows: p27 forward 5′-GAG TCA GCG CAA GTG GAA TTT C-3′ reverse 5′-GCG AAG AAG AAT CTT CTG CAG C-3′ and p57 forward 5′-GCC GGT CGA GGA GCA GAA TG-3′ reverse 5′-CCT GGA GGG ACG TCG TTC GA-3′ and GAPDH forward 5′-TGA TGA CAT CAA GAA GGT GAA G-3′ reverse 5′-TCC TTG GAG GCC ATG TAG GCC AT-3′.

B16-F10 melanocytes were treated with test samples for 48 hr. Total RNA was extracted using the TRI-Solution™ according to the manufacturer's instructions and quantified using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). cDNA synthesis was carried out from 1 µg RNA using AccuPower® PCR PreMix (Bioneer) following the manufacturer's recommendation. mRNA expressions of the MITF gene, tyrosinase gene, and TRP-1 were quantified using a Power SYBT® Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized with β-actin and fold change of expression was calculated with the ΔΔCT method. The primer sequences were as follows: mouse tyrosinase forward 5′-TACTTGGAACAAGCCAGTCGTATC-3′, reverse 5′-ATAGCCTACTGCTAAGCC CAGAGA-3′; mouse TRP-1 forward 5′- AAACCCATTTGTCTCCCAA -TGA-3′, reverse 5′-CGTTTTCCAACGG -GAAGGT A-3′, mouse MITF forward 5′-GGACTTTCCCTTATCCCATCCA-3′, reverse 5′-GCCGAGGTTGTTGGTAAAG -GT-3′. The PCR conditions were 95°C for 2 min followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min followed by a final 30 sec extension at 72°C. Data were analyzed using the Stepone™ software v2.3 (Applied Biosystems).