Fig 1: Evaluation of Hepatic Differentiation in HCC Cells after DNMT1 Knockdown(A) Expression of DNMT1 in clinical liver samples. The expression levels of DNMT1 in 20 primary HCC tumors and their corresponding paired non-tumorous tissues were determined by RT-qPCR. The Wilcoxon signed-rank test was used to evaluate the p value. DNMT1 and ALB protein levels in (B) epigenetically reconditioned and (C) DNMT1-knockdown HepG2 cells. Proteins were extracted 8 days after transfection. β-Tubulin was used as a loading control for immunoblots. Two distinct siRNAs were used to specifically target DNMT1 (siDNMT1_A and siDNMT1_B), and two scrambled siRNAs were used as negative controls (siCtrl_A and siCtrl_B). (D) CYP3A4 and CYP1A2 activity assay in the DNMT1-knockdown cells. Five days after transfection, HepG2 cells were treated with 25 μM dexamethasone for 72 hr, and the induced activities were measured. (E) Relative expression of ALB, NTCP, CYP3A4, and miR-122 following DNMT1 silencing in HepG2 and Hep3B cells. Gene expression levels were measured 6 and 8 days after transfection. (F) COBRA analysis of the ALB, NTCP, CYP3A4, and miR-122 genes after DNMT1 knockdown. Genomic DNA was extracted from HepG2 and Hep3B cells 6 and 8 days after cell transfection. Representative data from three COBRA experiments are shown for each gene. The data represent the mean ± SD. Statistical significance relative to the siCtrl-transfected cells: *p < 0.05; **p < 0.01; ***p < 0.001 (t test). M, methylated; U, unmethylated.
Fig 2: Differentiation Therapy by In Vivo Epigenetic Reconditioning: Hepatospecific Gene Expression and Methylation Profiles(A) Relative expression of the hepatic markers ALB, NTCP, CYP3A4, and miR-122 after epigenetic reconditioning in vivo. Total RNA and genomic DNA were extracted from resected tumors at the end of the treatment for analysis. (B) Scatterplots for Spearman’s rank correlation analysis between the gene expression levels and DNA methylation in the HCC tumors. The red and blue plots show the 5-AZA-treated mice (n = 8) and saline-treated mice (n = 8), respectively. (C) Relative expression levels of AFP, CDH1, G6Pc, TAT, CYP1A2, and HNF4A in HCC tumors after 5-AZA treatment. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001 (t test). Horizontal bars depict the average expression values.
Fig 3: Real-time RT-PCR of DSPP, BSP, and integrins on day 28. hDPSCs (passage number 4) were cultured in different substrate(s) in DMEM supplemented with 10% FBS for five days. Mineralization reagent including β-GP and AA was incorporated on day five. Dexamethasone (100 nM) (D2915, Sigma) was started to be added on day nine. The total culture time was 28 days. DSPP (a), BSP (b) and ITGA1 (c), ITGA4 (d), ITGA7 (e), and ITGB1 (f) mRNA expression was investigated. Different symbols represent significant differences, p < 0.01 (except for p < 0.05 between noncoated and Npnt-coated in (a); noncoated and BSA-coated and BSA-coated and FCOL1-coated in (b); noncoated and Fn-coated and noncoated and FCOL1-coated in (c); BSA-coated and FCOL1-coated in (d); noncoated and Fn-coated in (e); and noncoated and Npnt-coated in (f)) by post hoc Tukey's HSD test.
Fig 4: Hepatospecific Gene Expression and Drug-Metabolizing Activity in Epigenetically Reconditioned HCC Cells(A) Expression levels of four characteristic liver marker genes (ALB, SLC10A1, CYP3A4, and miR-122) in clinical samples. Boxplots illustrate the differential gene expression between 20 primary HCC samples (T) and their corresponding paired non-tumor tissues (NT). Mann-Whitney U tests were used to calculate p values. (B) Experimental design for evaluating liver gene expression and drug-metabolizing activity in HCC cells after epigenetic reconditioning. (C) Expression levels of selected hepatospecific genes in epigenetically reconditioned HepG2 and Hep3B cells. Total RNA was extracted after reconditioning with 2 μM 5-AZA for 12 days and 3 and 5 days of culture without 5-AZA (T1 and T2, respectively). The relative mRNA expression levels were determined by RT-qPCR. Non-reconditioned HCC cells were used as controls. (D) Relative levels of the AFP, CDH1, G6Pc, TAT, CYP1A2, and HNF4A mRNAs measured by RT-qPCR in the control and reconditioned HCC cells. (E) Evaluation of CYP3A4 and CYP1A2 enzyme activity. CYP activities were induced by treatment with 25 μM dexamethasone for 72 hr before assessment. All data shown in the figure represent the mean ± SD. Statistically significant differences in gene expression and CYP activity levels were achieved at *p < 0.05, **p < 0.01, and ***p < 0.001 (t test).
Fig 5: 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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