Fig 1: Pten loss suppressed cardiac development in vivo. a) A model for the generation of Pten conditional knockout mice. b) Immunohistochemistry experiment showing the expression of DNMT3B in the hearts of WT and Pten-/- mice. Scale bars, 20 µm. c) Quantification of the DNMT3B-positive cells in WT and Pten-/- hearts. Error bars indicate the mean ± SD (n = 5). p values were calculated by the Student's t-test: ***p < 0.001. d) The expression pattern of PTEN/AKT/DNMT3B signaling proteins in WT and Pten-/- hearts. e) Immunohistochemistry experiment showing the expression of IGF2 in the hearts of WT and Pten-/- mice at E12.5. Scale bars, 100 µm and 20 µm. f) Quantification of the IGF2-positive cells in WT and Pten-/- heart at E12.5. Error bars indicate the mean ± SD (n = 5). p values were calculated by the Student's t-test: ***p < 0.001. g) Immunofluorescence experiment showing the expression of NKX2-5 in the hearts of WT and Pten-/- mice at E18.5. Scale bars, 20 µm. h) Immunofluorescence experiment showing the expression of ISL1 and HAND2 in the hearts of WT and Pten-/- mice at E12.5. Scale bars, 50 µm. i–k) Quantification of the NKX2-5-positive, ISL1-positive, and HAND2-positive cells in WT and Pten-/- hearts. Error bars indicate the mean ± SD (n = 5). p values were calculated by the Student's t-test: **p < 0.01. l) A model of how Pten deletion suppresses cardiomyocyte differentiation. During cardiomyocyte differentiation from ESCs, Pten suppresses the expression of the Dnmt3 family; the loss of Pten promotes the non-CG methylation of cardiomyocyte genes and Igf2, which leads to the inhibition of cardiomyocyte differentiation.