Treatment with oxamflatin resulted in decreased global DNA methylation levels of two-cell-stage porcine SCNT embryos through downregulation of DNMT1. (A) Immunofluorescence co-staining of global 5-mC and 5-hmC between C-NT and T-NT groups at the two-cell and four-cell stages. The experiments were repeated three times and each replication included at least 10–15 embryos. Representative examples are shown. Scale bar, 20 μm. (B) Quantification of mean fluorescence signal intensities of 5-mC and 5-hmC between C-NT and T-NT groups. (C) The relative mRNA expression levels of DNMT1, DNMT2, DNMT3a, and DNMT3b in MII oocytes. (D and E) The relative mRNA expression levels of DNMT1 at the two-cell- and four-cell-stage embryos between C-NT and T-NT groups, respectively. The relative expression level of mRNA was calculated with β-actin. T-NT, SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation; C-NT, nontreated SCNT embryos. Data were presented as mean±standard deviation (SD). (*) p<0.05; (**) p<0.01.