Fig. 5. Melatonin upregulates DNMT1 via MT1/ERK/c-MYC axis.

A The cells were treated with actinomycin D (500 ng/ml) 30 min before treating melatonin for 12 h. The FKBP52 mRNA expression was analyzed by real-time PCR. n = 5. B, K SH-SY5Y cells were treated with melatonin (1 μM) for 30 min and then with cortisol (1 μM) for 12 h. B The mRNA expression of epigenetic-regulated genes was analyzed by real time PCR. n = 5. C SH-SY5Y cells were treated with melatonin for 30 min and then with cortisol for 24 h. D Mice were injected with melatonin (10 mg/kg) and then with corticosterone (10 mg/kg) for 7 days. C, D DNMT1 expression was detected by western blot. Loading control is β-actin. n = 5. E, F, H SH-SY5Y cells were treated with melatonin for 12 h. E The MT1 and MT2 mRNA expression were analyzed by real time PCR. n = 5. F MT1 and MT2 levels were detected by western blot. Loading control is β-actin. n = 5. G The cells were transfected with NT or MT1 siRNA 24 h before melatonin treatment for 24 h. FKBP4 levels were detected by western blot. Loading control is β-actin. n = 5. H The cells were immunostained with MT1 (green), Gαq (red) and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. I The cells were transfected with NT or MT1 siRNA 24 h before melatonin treatment for 12 h. The p-ERK and ERK levels were investigated by western blot. Loading control is β-actin. n = 5. J PD98059 (50 μM) was treated 30 min before melatonin treatment for 12 h. The p-c-MYC and c-MYC levels were detected by western blot. Loading control is β-actin. n = 5. K The cells were immunostained with c-MYC (green) and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. All blots and immunofluorescence images are representative. The representative images were acquired by SRRF imaging system. All data are presented as a mean ± S.E.M. *p < 0.05 versus control or control + NT siRNA, ^#p < 0.05 versus melatonin or melatonin + NT siRNA.