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. 2018 Apr 10;9:1373. doi: 10.1038/s41467-018-03714-x

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© The Author(s) 2018

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. 1 — NKX2-5 regulates cardiomyocyte differentiation. a Schematic representation of NKX2-5^eGFP/w and NKX2-5^−/− (NKX2-5 null) genotype. b Immunofluorescent detection of NKX2-5, ACTN2 and GFP in NKX2-5^eGFP/w and NKX2-5^−/− cultures at day 14 of cardiac differentiation. Nuclei counterstained with DAPI. Scale bar = 50 μM. c Bar graph quantifying GFP and ACTN2 expression in differentiating NKX2-5^eGFP/w and NKX2-5^−/− cultures, as determined by flow cytometry (see Supplementary Fig. 1). Data represent mean ± SEM (n = 5). **p < 0.01 (Student’s t-test). d Q-PCR analysis of NKX2-5^eGFP/w and NKX2-5^−/− cultures at day 14 of differentiation. NKX2-5 null cardiomyocytes show normal expression of characteristic cardiomyocyte markers. Data represent mean ± SEM (n = 4). *** p < 0.001 (Student’s t-test). e, f Representative flow cytometry plots (e) and bar graph (f) of PDGFRα expression in NKX2-5^eGFP/w and NKX2-5^−/− cultures at day 42 of differentiation. Numbers on plots are percentage of cells in quadrant. Data represent mean ± SEM (n = 4). ***p < 0.001 (Student’s t-test). g, h Representative flow cytometry plot at day 14 of differentiation (g) and bar graph (h) of a time course of VCAM1 expression in differentiating NKX2-5^eGFP/w and NKX2-5^−/− cultures. Numbers on plots are percentage of cells in quadrant. Data represent mean ± SEM (n = 4). ***p < 0.001 (Student’s t-test)