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. 2017 Jun 9;7:3136. doi: 10.1038/s41598-017-03475-5

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© The Author(s) 2017

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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Npnt is essential for COUP-TFII silencing mediated acceleration of myogenesis. (A) Two days post-differentiation, indicated cells were fixed and immunostained with pan-MHC (left panel), and their fusion rates were then quantified as the percentage of MyHC+ nuclei versus total nuclei (right panel). (B) Quantitative RT-PCR examination of indicated genes expression; (C) Immunoblotting of indicated protein expression in control, COUP-TFII silencing, Npnt silencing and double silencing C2C12 cells, one day post-differentiation. Cells were pre-silencing of indicated genes 3 days and reseeded before serum withdrawal. (D) COUP-TFII overexpressed cells were infected with control or Npnt overexpressed lentiviruses for three days and the serum was withdrawn for differentiation. Six days post-differentiation, indicated cells were fixed and immunostained with pan-MHC antibodies. (E) Illustration of the molecular mechanisms of COUP-TFII-mediated inhibition of myoblast fusion. Data are shown as mean ± SEM. Two-tailed Student’s t test; **p < 0.01, ***p < 0.001.