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. 2019 Aug 6;9:11360. doi: 10.1038/s41598-019-47851-9

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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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Effects of apical hyperosmolarity on cell viability. Assays were performed after treatment during 2- or 24-h with hyperosmotic media or isosmotic control medium. (A) Number of adhering cells measured by cell counting. (B) Cell viability estimated by measuring LDH released in the medium before and after 1% triton treatment and expressed as ratio of alive and total cells. (C) Cell viability evaluated with MTT test, and (D) ADP/ATP cell ratio from the amount of ATP and ADP cell content (expressed as relative luminescence units in the curly bracket). In (A) and (B) experiments, cells were plated on 24-well plates and grown for 3 days before hyperosmotic stress assay. In (C) and (D) experiments, cells were plated on 96-well plates and grown 3 days before hyperosmotic stress assay. Values are from three to four independent experiments (n = 8–32 for each experimental group). Mean significant differences (P < 0.05) are indicated by a different letter. Main factor and interaction effects are indicated with *P < 0.05, **P < 0.01 and ***P < 0.001. NS: Non-significant difference.