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. 2018 Jun 22;8:9542. doi: 10.1038/s41598-018-27854-8

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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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Expression of SMURF1 during human heart development. (A) SMURF1 expression in human embryonic, fetal and adult hearts. Expression level was examined using qRT-PCR and normalized to average values of three housekeeping genes (GAPDH, COX4 and ATP6). Expression level is shown as fold change compared to expression in the adult heart. Variations between groups were compared using a one-way ANOVA. *p < 0.05, ***p < 0.001, ****p < 0.0001. (B) IHC staining of SMURF1 in a human heart section from 35 dpf. b’ and b” enlargement of boxes indicated in B; closed arrow heads show endothelial cells, open arrow heads show membrane localization and red arrow shows nuclear localization (see Supplemental Fig. 1 for enlargement). (C) IHC staining of SMURF1 in a human embryonic heart section from 38 dpf. c’ Enlargement of the boxed area in C. c” IF-IHC image of a neighboring section in the area marked by the box in C (SMURF1 (red), TBX2 (green); nuclei are stained with DAPI (blue). c”’ enlargement of box indicated in c”. (D) IHC staining of SMURF1 in a human embryonic heart from 40 dpf. Red outline: condensed CNC mesenchyme, green outline: patches of SMURF1 positive cells in the coronary sinus. Asterisk indicates the primary atrial septum. (E) SMURF1 staining in human embryonic heart from 49 dpf. Closed arrow heads show remnants of condensed CNC-derived mesenchyme, open arrow head marks the coronary vessel. (F) SMURF1 staining in human embryonic heart from 55 dpf. Asterisk marks area of sinus atrial node and open arrows mark coronary vessels. f’ IF-IHC of SMURF1 in a neighboring section showing SMURF1 expression (red) in an the boxed area in F. (G) SMURF1 staining in human embryonic heart from 70 dpf. Arrows mark SMC-positive areas. Abbreviations: A: Atrium, Ao: Aorta, AoR: Aortic root, AVN: Atrioventricular node, CS: Coronary sinus, Inf: Infundibulum, LA: Left atrium, LSCV: Left superior caval vein, LV: Left ventricle, OFT: Outflow tract, PC: Parietal cushion, Pt: Pulmonary trunk, RA: right atrium, RV: Right ventricle, SC: Septal cushion, SVC: Superior vena cava, TCV: Tricuspid valves, V: Ventricle.