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. 2018 Apr 4;8:5618. doi: 10.1038/s41598-018-23918-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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Precocious differentiation rescue in Lmna^−/− MB by Smad6 inactivation. (A) Quantification of Smad6 expression by RT-qPCR in presence of scramble siRNA (SiCtrl) or a SiRNA against Smad6. Values are relative expression normalized on Hprt1 housekeeping gene expression. (B) Smad6 protein levels quantified by western blot in presence of SiCtrl or SiSmad6. Tubulin expression levels are provided as normalizer. (C) Immunofluorescence assay showing MyHC (in green) and DAPI (in blue) of wild-type and Lmna^−/− H-2K myotubes after 24 hours of treatment with Smad6-SiRNA (or SiCtrl) and recombinant Bmp4 (100ng/mL) (or vehicle) followed by 48 hours of differentiation. Scale bar: 200 µm. (D) Differentiation index (percentage of nuclei in MyHC positive cells) for each condition showing that the double treatment by Smad6-SiRNA and recombinant Bmp4 in Lmna^−/− H-2K MB is able to rescue the phenotype and to prevent the observed premature differentiation. At least 350 nuclei per conditions were analyzed. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Full-length blots are presented in Supplementary Figure 6.