Fig 1: Reserpine treatment in SH–SY5Y cells induced decreased DNA methylation levels. (A,B) Reserpine treatment resulted in elevated protein expression of SNCA in SH–SY5Y cells (n = 3 for each group, one-way ANOVA with Dunnett post hoc test, *p < 0.05 vs. control). (C,E) Reserpine treatment decreased DNA methylation of SNCA for a subset of CpG by pyrosequencing analysis. (C,E) are representative samples and statistical analysis, respectively (n = 6 for each group, student t-test, **p < 0.01 vs. control). (D,F) Reserpine treatment did not change the DNA methylation levels of ATG3. (D,F) are representative samples and statistical analysis, respectively (G) Differential expression analysis for methylation-related enzymes by qRT-PCR (n = 5 for each group, student t-test, *p < 0.05, **p < 0.01 vs. control). (H,I) Reserpine treatment resulted in decreased protein expression of DNMT3b in SH-SY5Y cells (n = 3 for each group, student t-test, * *p < 0.05 vs. control). All data are expressed as mean ± SEM; Con, control; RES, reserpine.
Fig 2: Methylation Profiles of Liver Marker Genes in Reconditioned Cells(A) Comparison of the methylation levels of ALB, SLC10A1, CYP3A4, and miR-122 between human hepatocytes and HCC cells. COBRA was performed to evaluate the CpG methylation ratios (%) of gene promoter regions. The CpG sites were identified by in silico analysis (Figure S1). (B) Effect of 5-AZA treatment on the global DNA methylation level in HepG2 cells. The levels of 5'-methyl-2'-deoxycytidine (5-MedCyd) in the DNA samples were quantified with an ELISA-based method at the indicated times. (C) Methylation status of the ALB, SLC10A1, CYP3A4, and miR-122 genes in reconditioned HepG2 and Hep3B cells, as evaluated by COBRA. (D) Methylated DNA levels after selective knockdown of DNMT1, DNMT3A, and DNMT3B in HepG2 cells. The histograms show the 5-MedCyd ratios from siDNMT-transfected cells relative to those of cells transfected with control siRNAs. Histograms shown in the figure represent the mean ± SD. Statistical significance versus control cells (non-reconditioned and siCtrl-transfected cells): *p < 0.05; **p < 0.01; ***p < 0.001 (t test). Representative data from three COBRA experiments are shown for each gene. M, methylated; U, unmethylated.
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