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. 2018 Feb 8;8:2644. doi: 10.1038/s41598-018-20781-8

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© The Author(s) 2018

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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Physiological properties of cells under chronic heat stress in wild-type and ubi4Δ cells. (a) Phloxine B staining. Wild-type W303 and ubi4Δ cells grown at 25 °C were transferred to 40.5 °C, or wild-type cells incubated at 65 °C for 10 min and Phloxine B-stained cells were observed by fluorescence microscopy. Dead cells were stained by Phloxine B. (b) Quantification of Phloxine B-unstained cells in (a). Date are presented as the mean value of three independent experiments, except for the value of ubi4Δ cells at 40.5 °C for 24 h, which is an average of two experiments. Standard errors (SE) are shown. (c–e) Lyp1-3HA, Hxt3-3HA and ubiquitin accumulations in wild-type and ubi4Δ cells at the indicated times after exposure to 40.5 °C. Anti-HA antibody for Lyp1-3HA and Hxt3-3HA. Anti-phosphoglycerate kinase (PGK) antibody as a control for protein loading. Molecular weight markers are indicated. The levels of ubiquitinated proteins were lower in ubi4Δ cells than in wild-type cells after 3 h at 40.5 °C.