Transfection with small interfering RNA (siRNA) against Nrf2

Nrf2 siRNAs were purchased from Santa Cruz Biotechnology Inc., (Dallas, TX, USA), NRF2-siRNA, 5′-UGAAAGCACAGCAGAAUUTT-3′ and control-siRNA, 5′-GAGCGGCCGAGCAACGUCUAU-3′. According to the manufacturer's protocol, the siRNAs (final siRNA concentration, 10 nM) were transfected into B16-F10 cells by using the Lipofectamine^® RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells were seeded in 6-well culture plates and incubated with Nrf2 siRNA at 50 nM for 6 h in serum-free OPTI-MEM media (Invitrogen; Thermo Fisher Scientific Inc.). Following incubation for 24 h at 37°C, the transfected cells were used for subsequent experiments.