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. 2017 Jul 3;7:4543. doi: 10.1038/s41598-017-04876-2

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© The Author(s) 2017

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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Hyperosmolarity does not activate autophagy through MTOR-AMPK-ULK1 axis in NP cells. (a) Western blot analysis of NP cells treated with increasing osmolarity (330–600 mOsm/kg H₂O) showed that the levels of pULK1 Ser757 and pULK1 Ser777 were not affected by hyperosmolarity. (b, c) Densitometric analyses of multiple Western blots represented in (a) confirmed lack of effect on ULK1 phosphorylaiton at Ser757 and Ser777 by hyperosmolarity. (d) Western blot analysis of NP cells cultured in hyperosmotic media for increasing lengths of time demonstrated that phosphorylation of ULK1 at both Ser757 and Ser777 by MTOR and AMPK respectively was not affected by hyperosmolarity till 72 h. (E, F) Denstiometric analyses of multiple Western blots represented in (d). Bars represent mean ± SEM (n = 3). One-way ANOVA with Sidak’s multiple comparisons test was used to determine statistical significance. NS, non-significant. Western blot images were cropped and acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images.