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Fig. 3 | Cell Regeneration

Fig. 3

From: CTCF acetylation at lysine 20 is required for the early cardiac mesoderm differentiation of embryonic stem cells

Fig. 3

The CTCF-K20 mutation impaired the cardiac differentiation of embryoid bodies. (A) Chromatogram from Sanger sequencing showing the sequence of the mutated DNA. (B) Western blots showing CTCF, CTCF-K20ac and β-ACTIN levels in both WT and CTCF-K20R mESCs. (C) RT–qPCR analysis of the expression of pluripotency (Oct4 and Nanog), ectoderm (Nestin and Olig2), endoderm (Sox17 and Afp), and cardiac mesoderm (Tbx5b and Myl7) marker genes using RNA lysates from WT and CTCF-K20R mESCs and EBs at day 12 after mESC differentiation. Expression was normalized to Gapdh. (D) Semiquantitative analysis of beating EBs. The data are presented as the averages of three experiments. (E) RT–qPCR analysis of the expression of selected genes (Tnnt2, Nkx2-5) in WT and CTCF-K20R ESCs and EBs on day 12. (F) Immunostaining analysis of the cardiomyocyte marker cTnT after EB differentiation on day 12. The scale bar represents 100 μm. (G) Scatter plot showing DEGs between WT and CTCF-K20R EBs 4 days after mESC differentiation. Two independent RNA-seq experiments were performed for each sample. (H) GO analysis of the significantly downregulated genes shown in Fig. 3G. (I) Heatmap showing the expression of marker genes related to myocardial differentiation that were derived from a previous report (Cunningham et al. 2017). (J) RT–qPCR analysis of the selected genes in both WT and CTCF-K20R mESCs and EBs at day 4 after mESC differentiation. Bar graphs in (C), (D), (E) and (J) represent the mean ± s.d. (n = 3) and P values were determined by t test (**P < 0.01, ***P < 0.001)

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