Fig. 4
Regulatory effect of melatonin on hypermethylation of Muc2 promoter induced by rVvpM. a Genomic DNA treated with rVvpM for 12 h was prepared. Time responses of rVvpM in methylation status of Muc2 gene at − 274 and − 193 CpG sites were determined by methyl-specific PCR (MSP) analysis. The relative level of Muc2 methylation is shown, compared to the unmethylated form. Data represent means ± S.E. n = 4. *, p < 0.05 vs. 0 h. b Cells were transfected with siRNA for PKCδ or ERK1/2 for 24 h, or pre-treated with NAC (10 μM) or 5-aza (1 μM) for 30 min prior to rVvpM exposure for 12 h. The relative level of Muc2 methylation is shown, compared to the unmethylated form. Data represent means ± S.E. n = 4. *, p < 0.05 vs. nt siRNA. #, p < 0.01 vs. rVvpM + nt siRNA. c Cells transfected with siRNA for MT2, Gαq, or NCF-1 for 24 h were incubated with melatonin (1 μM) for 30 min prior to rVvpM exposure for 12 h. The relative level of Muc2 methylation is shown, compared to the unmethylated form. Data represent means ± S.E. n = 4. *, p < 0.05 vs. nt siRNA. #, p < 0.01 vs. rVvpM + nt siRNA. $, p < 0.05 vs. rVvpM + melatonin+ nt siRNA. d Cells were pre-treated with 5-aza (1 μM) for 30 min prior to rVvpM exposure for 24 h. The level of Muc2 production was quantified by ELISA. Data represent means ± S.E. n = 4. *, p < 0.01 vs. vehicle. #, p < 0.05 vs. rVvpM alone