Fig. 3

Upregulation of TET2 during in vitro and in vivo cardiac differentiation. A-C qRT-PCR analysis of the relative TET2 mRNA level during 12-day cardiac differentiation in hESCs-CMs A, hiPSCs-CMs B and mESCs-CMs C (n = 3–4). D qRT-PCR analysis of the relative TET2 mRNA level in embryonic and neonatal heart samples (E17.5, P2 and P9) from wild-type C57BL/6 mice (n = 5). E and F Western blot analysis E and quantification F of the relative TET2 protein level during 12-day cardiac differentiation of hESCs (n = 3). G and H Western blot analysis G and quantification H of the relative TET2 protein level in embryonic and neonatal heart samples (E17.5, P2 and P9) from wild-type C57BL/6 mice (n = 3). I Flow cytometry analysis of TET2-positive cells during 12-day cardiac differentiation of hESCs (n = 3). J Immunofluorescence of TET2 and 5hmc in 12-day cardiac-differentiated hESCs (Scale bar = 10 μm). K Dot blot analysis of global 5hmC level during 12-day cardiac differentiation of hESCs. Methylene blue (MB) staining demonstrated equal loading. D0, D3, D6, D9 and D12 indicated the time points at Day 0, Day 3, Day 6, Day 9 and Day 12, while E17.5, P2 and P9 indicated Embryonic day 17.5, Postnatal day 2 and 9, respectively. Quantitative data were presented as mean ± SEM, while statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as *P < 0.05, **P < 0.01 and ***P < 0.001