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. 2021 Feb 11;11:3588. doi: 10.1038/s41598-021-82954-2

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Introduction of Smad4 rescues the nuclear localization of Nkx2-5 in mutant cells and depletion of the Smad4 gene blocks the nuclear translocation of Nkx2-5 in fibroblast cells. (A) Double immunohistochemistry with anti-Nkx2-5 (red) and anti-Flag (green) antibodies. Cells from Smad4-cKO embryonic hearts were transfected with an empty vector (pDEF) as a control (upper panels) or Flag-tagged Smad4 expression vector (lower panels). Arrows show the failure of Nkx2-5 to localize to the nucleus. DAPI was used to stain nuclei. (B) Percentage of cells showing nuclear localization of Nkx2-5. Nuclear localization of Nkx2-5 was significantly increased in the presence of Smad4, compared to cells transfected with empty vector. Data were collected from 20 slides for each experiment (n = 4 and 4 for control and Smad4-cKO, respectively). Data are presented as mean ± SEM. *P < 0.01 (Mann–Whitney U test). (C) Double immunohistochemistry with anti-Myc (red) and anti-Smad4 (green) antibodies. Fibroblast cells from Smad4f.^/f embryos were transfected with the Myc-Nkx2-5 expression vector and showed nuclear localization of Nkx2-5. After depleting Smad4 by transfecting fibroblasts with Cre expression vector twice, Nkx2-5 was no longer localized to the nucleus. DAPI was used to stain nuclei. (D) The proportion of non-nuclear localization of Nkx2-5 in control (transfected with Myc-Nkx2-5) and Smad4-cKO (transfected with Myc-Nkx2-5 after two rounds of Cre expression vector transfection) cells. Data were collected from 20 slides for each experiment (n = 3 and 3 for control and Smad4-cKO, respectively). Data are presented as mean ± SEM. **P < 0.01 (Mann–Whitney U test).