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. 2018 Jun 14;8:9101. doi: 10.1038/s41598-018-27414-0

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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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Small interfering RNA (siRNA)-mediated silencing of WNK1, but not SPAK/OSR1 kinases, induced myotube atrophy and increased atrogene expression. (A) Immunoblots showing the siRNA knockdown efficiency of WNK1, SPAK, or OSR1 in C2C12 cells. (B) After C2C12 cells were pre-treated with siRNA, the cells were incubated in differentiation medium for 4 days, and immunofluorescence study was performed with a myosin heavy chain (MHC) antibody. Scale bar, 200 μm. (C) Histograms showing proportions of myotubes according to the diameters. The proportions of atrophic myotubes were markedly higher in the WNK1-silenced group (n = 5 or 6 per experimental group). (D) The mean myotube diameter value in the WNK1-silenced group was lower than that in the control group (n = 5 or 6 per experimental group). (E) The fusion index represents the percentage of multinucleated MHC-positive myotubes per vision field after 96 h of differentiation (n = 5 or 6 per experimental group). Wnk1-targeted siRNA suppressed the myogenic fusion of myoblasts into myotubes. (F) Quantification of MAFbx, MuRF1, Bim, Noxa, and bcl6 in siRNA-treated C2C12 cells by real-time polymerase chain reaction analysis (n = 4 per experimental group). mRNA levels were normalized against those of β-actin. Values are presented as the mean ± standard error of the mean. *P < 0.05; **P < 0.01. Small interfering RNA, siRNA; WNK, with-no-lysine (K); SPAK, SPS/STE20-related proline-alanine–rich kinase; OSR1, oxidative stress responsive 1; MHC, myosin heavy chain; MAFbx, muscle atrophy F-box; MuRF1, Muscle RING-finger protein-1. Full-length blots are presented in Supplementary Fig. 8.