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. 2023 Jul 14;9:244. doi: 10.1038/s41420-023-01548-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A The integrating strategy of the Zhx1 gene in the Rosa26 locus via CRISPR/Cas9 technology. B, C The mRNA (B) and protein levels (C) of Zhx1 overexpression. D The expression of pluripotent genes (Oct4, Sox2, Nanog, and Esrrb) after Zhx1 overexpression. E The expression of epiblast genes (T, Fgf5, and Dnmt3b) on day 2 of cardiomyocyte differentiation after Zhx1 overexpression. F The expression of mesoderm genes (Flk1, Pdgfrα, Eomes, Mixl1, and Mesp1) on day 4 of cardiomyocyte differentiation after Zhx1 overexpression. (G) The percentage of Flk1⁺/Pdgfrα⁺ cells on day 4 of cardiomyocyte differentiation after Zhx1 overexpression. H, I The expression of the CP genes (Nkx2.5, Isl1, Mef2c, Tbx5, Hand1, and Hand2) (H) and cardiomyocyte markers (cTnT, Myl2, Myl7, α-MHC, β-MHC, and Slc8a1) (I) after Zhx1 overexpression during cardiomyocyte differentiation. J The western blot results for the expression of cTNT and α-Actinin at day 8 of cardiomyocyte differentiation after Zhx1 overexpression. K The expression of cTNT protein after Zhx1 overexpression during cardiomyocyte differentiation through immunofluorescent staining. L The percentage of cTNT⁺ cardiomyocytes after Zhx1 overexpression. M The percentage of beating EBs on day 8 of EB differentiation after Zhx1 overexpression. N The expression of cardiomyocyte markers after Zhx1 overexpression on day 8 of EB differentiation. O The expression of cTNT protein after Zhx1 overexpression during the EB differentiation. Scale bar, 100 μm. Data are presented as the mean ± SEM (n = 3). The statistical significance is performed according to Student’s t-tests (unpaired two-tailed). *p < 0.05, **p < 0.01, and ***p < 0.001 versus 46C-Ctrl.