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. 2020 Sep 23;10:15511. doi: 10.1038/s41598-020-72613-3

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Osmolyte dose-dependent effects on HSP70 expression and distribution of mHtt in the diffusible ensemble (diffuse) versus inclusion body (IB) formats. The 14A2.6 line of PC12 cells were plated in 96 well plates. Ponasterone (PA) was added to a final concentration of 5 μM at plating (t = 0) to induce mHtt expression. Osmolytes (sorbitol, trehalose, urea, and trehalose + urea) were added at t = 24 h to final concentration of 25, 50, 100 and 150 mM as indicated and continue to incubate at 37 °C for another 24 h. The timeline of the experiment is as shown in Fig. 1. (A) Representative images of cells under the specified conditions. The scale bar is of 50 µm. (B) Quantitation of HSP70 of cells treated with specified concentrations of osmolytes. (C) Total IB count per unit cell. D. % distribution of mHtt signal in IB versus diffuse formats. Probability of difference P > 0.05 is defined as not significant, between 0.01 and 0.05 is significant (*), < 0.01 is very significant (**), and < 0.001 is extremely significant (***).