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. 2011 Aug 16;39(21):9194–9205. doi: 10.1093/nar/gkr658

Figure 4.

Figure 4.

PCNA and DMAP1 domains of DNMT1, but not its catalytic region, are directly involved in the regulation of E-cadherin expression and nuclear translocation of β-catenin in DNMT1ΔE3−6 cells. (A) Immunolocalization analyses showing re-expression of E-cadherin and relocalization of β-catenin from the cell nucleus to newly formed cell–cell contacts after reintroduction of full-length DNMT1 in DNMT1ΔE3−6 cells. At least three stable clones or transient transfections were analyzed. Bar: 10 μm. (B) Upper panel: Semiquantitative analysis of mRNA expression by RT–PCR of exons 1–6 and exons 32–34 of the DNMT1 gene in DNMT1ΔE3−6 cells transfected with full-length DNMT1 cDNA (DNMT1), or deletion forms of DNMT1 cDNA lacking the catalytic domain (DNMT1ΔCat), or the catalytic and BAH domains (DNMT1ΔCat/BAH). Results are representative of two different experiments. Actin expression was used as a loading control. Lower panel: Quantitative analysis of E-cadherin mRNA expression by qRT–PCR in DNMT1ΔE3−6 cells transfected with full-length DNMT1 cDNA (DNMT1), or deletion forms of DNMT1 cDNA lacking the catalytic domain (DNMT1ΔCat), or the catalytic and BAH domains (DNMT1ΔCat/BAH). Relative expression levels are normalized to actin expression. Results are representative of three different experiments and the mean ± SD are shown. **P < 0.001. (C) Semiquantitative analysis by immunoblot of E-cadherin, β-catenin and β-catenin-ABC protein expression in DNMT1ΔE3−6 cells transfected with full-length DNMT1 cDNA (DNMT1), or deletion forms of DNMT1 cDNA lacking the catalytic domain (DNMT1ΔCat), or the catalytic and BAH domains (DNMT1ΔCat/BAH). Results are representative of two different experiments. β-tubulin expression was used as a loading control. (D) Normalized Luciferase/Renilla activity of reporter vector containing multimerized promoter sequences recognized by β-catenin-lef/tcf complexes in DNMT1ΔE3−6 cells transiently transfected with full-length DNMT1 cDNA (DNMT1), or deletion forms of DNMT1 cDNA lacking the catalytic domain (DNMT1ΔCat), or the catalytic and BAH domains (DNMT1ΔCat/BAH). Analyses were performed in triplicate and the mean ± SD are shown. **P < 0.001.