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. 2018 Nov 16;8:16957. doi: 10.1038/s41598-018-35150-8

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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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DUX4 inhibits myogenic differentiation. (A) Immunofluorescence for myosin heavy chain (MHC, MF20, red) on LHCN-M2-iDUX4 cells after 2 days of differentiation in the presence of 3.1 and 12.5 ng/mL doxycycline. Nuclei were counterstained with DAPI (blue). Scale bar 100 µm. (B) RT-qPCR for MYOD1, MYF5, MYOGENIN, DESMIN, MYH3 (embryonic myosin heavy chain) on LHCN-M2-iDUX4 cells presented in (A) Data are presented as mean ± SEM; ***p < 0.001, ****p < 0.0001, T-test. Gene expression values are presented as fold difference to GAPDH (n = 4). (C) Western blot for DUX4 and MYOD1 on LHCN-M2-iDUX4 cells induced with various doses of doxycycline (dox) over 14 hours (left), and with 200 ng/mL doxycycline for 2 or 14 hours. (D) RT-qPCR for DUX4, MYOD1 and MYF5 on LHCN-M2-iDUX4 cells induced for 14 hours with various concentrations of doxycycline (ng.mL). Data represents mean ± SEM; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 by one-way ANOVA with Tukey’s post hoc test. Results are presented as fold difference compared to GAPDH (n = 3).