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. 2019 Dec 2;9:18091. doi: 10.1038/s41598-019-54574-4

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© The Author(s) 2019

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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A pulse of BIO improves skeletal muscle differentiation in-vitro. (A) Schematic representation of C2C12 treatment: C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and then exposed to either GM or DM for 96 h. (B) Immunofluorescence analysis of differentiation marker MHC (green) in C2C12 cells. Nuclei are counterstained with DAPI dye (blue). (C) The histograms indicate the fusion index and the myotube size of all samples at day 4 of myogenic differentiation. The fusion index was calculated as the ratio of the number of nuclei inside myotubes (MHC positive cells) to the number of total nuclei at day 4 of myogenic differentiation. A myotube was defined by the presence of at least three nuclei within a continuous cell membrane. Data represent means ± SEM relative to GM or DM treated cells Data were analyzed by one-way analysis of variance ANOVA: (C) left panel (ns), (C) right panel (***P < 0.001). (D) Data of myotubes size are expressed as mean diameter in micrometers of ten myotubes measured per field. Bar scale = 100μm.Data (collected from four independent experiments) represent means ± SEM relative to GM or DM treated cells. Data were analyzed by one-way analysis of variance ANOVA (***P < 0.001).