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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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APN accumulation and exosome secretion. (A,B) Differentiation dependence of APN-accumulation and exosome secretion. (A) Proteins in cell lysates. Experimental scheme was summarized. Blots for T-cad (130 kDa and 100 kDa), APN (30 kDa) and α tubulin (48 kDa) were shown. Data are mean ± s.e.m., n = 3, n.s; not significant; **P < 0.01; ***P < 0.001; Tukey’s test. (B) Undifferentiated myoblasts (Day 0) and differentiated myocytes (Day 4) of C2C12 cells were treated with or without purified APN for 48 hrs. Secreted exosomes were purified and proved by antibodies against typical exosome cargos indicated. mean ± s.e.m., n = 3. *P < 0.05. (C–E) T-cad dependent APN-accumulation and exosome secretion. (C) Cell lysates of C2C12 cells transfected with siRNA for T-cad or negative control. mean ± s.e.m., n = 3. ***P < 0.001. (D) Cell lysates of C2C12 cells transfected with siRNA for AdipoR1/AdipoR2 or negative control. mean ± s.e.m., n = 3. n.s; not significant. mRNA knockdown levels were shown. mean ± s.e.m., n = 3. (E) siRNA for T-cad or negative control were transfected into C2C12 cells. Differentiating cells (Day 2) were treated with or without purified adiponectin for 48 hrs. Secreted exosomes were purified and proved by antibodies against typical exosome cargos indicated. mean ± s.e.m., n = 3. *P < 0.05.