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. 2011 Feb 8;6(2):e16702. doi: 10.1371/journal.pone.0016702

Figure 1. DNA re-methylation after 5-Aza-dC treatment.

Figure 1

(A) Schematic outline of the experimental design. SMMC-7721 cells were treated with 5-Aza-dC at 10 µM for 4 days to induce global demethylation. At Day 4, 5-Aza-dC was removed and cells were replenished with normal culture medium (DMEM-high glucose, supplemented with 10% FBS). 5-Aza-dC treated cells were allowed to recover in the absence of 5-Aza-dC for an additional 4 weeks. DLC1 mRNA (B) and protein (C) expression in SMMC-7721 was gradually re-silenced when released from 5-Aza-dC treatment at Day 4. (D) Consistent with re-silencing of DLC1 expression, SMMC-7221 cells gradually re-acquired DLC1 promoter methylation after removal of 5-Aza-dC treatment, as revealed by methylation-specific PCR. MSP: methylation-specific PCR, USP, unmethylation-specific PCR. Semi-quantitative analysis was done by AlphaEaseFC software (Alpha Innotech, San Leandro, CA). (E) In addition to DLC1, gene re-silencing was also observed in multiple hypermethylated genes, including E-cadherin, GSTP1, uPA and KRT19. In contrast, the expression level of unmethylated tumor suppressor gene p16CDKN2A was not affected and remained constant throughout.