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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 upregulate the levels of canonical autophagic markers. (a) Western blot analysis of NP cells cultured under increasing osmolarity (330–600 mOsm/kg H₂O) showed that the levels of LC3-II, SQSTM1, ATG12-ATG5, and BECN1 did not change by hyperosmolarity. However, TonEBP expression increased under hyperosmotic condition. (b–d) Densitometric analyses of multiple Western blots represented by (a) confirmed significant induction of TonEBP, while LC3-II and SQSTM1 levels remained unaltered (n = 5). (e) Western blot analysis of NP cells cultured under hyperosmotic condition for increasing lengths of time demonstrated that LC3-II, SQSTM1, ATG12-ATG5, and BECN1 levels were unaffected by hyperosmolarity up till 72 h. (f–i) Densitometric analyses of multiple Western blots shown in (e) (n = 3). Bars represent mean ± SEM. 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.