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. 2019 Jan 9;9:16. doi: 10.1038/s41598-018-37115-3

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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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Intracellular distribution of APN. Confocal immunofluorescence micrographs of C2C12 cells. (A–C) Cells at indicated induction periods were stained with anti-adiponectin (APN; red), anti-CD63 (MVB; green). Cell nuclei were counterstained with DAPI (Nuclei; blue). Scale bars, 100 μm. (A) Undifferentiated C2C12 myoblasts. Scale bars, 100 μm. (B) Differentiating C2C12 myocytes. Scale bars, 100 μm; 30 μm (higher magnifications). Overlap coefficient of APN and CD63 in differentiating myocytes was calculated as described in Methods. Data are mean ± s.d., n = 5. (C) Fully differentiated C2C12 myotubes. arrow heads; differentiating myocytes. Scale bars, 200 μm; 100 μm (higher magnifications). (D) Differentiating C2C12 cells treated with siRNAs. siRNA for T-cad, AdipoR1/AdipoR2 or negative control were transfected into C2C12 cells. Differentiating cells (Day 2) were treated with or without purified adiponectin for 24hrs. Cell nuclei were counterstained with DAPI (Nuclei; blue). Anti-CD63 (MVB; green). Anti-adiponectin (red) for left panels and Anti-T-cadherin (red) for right panels. Scale bars, 100 μm.