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. 2020 Jun 18;10:9926. doi: 10.1038/s41598-020-66568-8

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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 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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Hypoxia and hypoxia followed by re-oxygenation alter RhoA-ROCK and MAPK signalling and response to CA4P in endothelial cells. (a) Confluent endothelial cells were exposed to varying oxygen levels for 14 h and then CA4P (1 μM) or vehicle (PBS) were added for a further 15 min. Cells were analysed for the expression and/or phosphorylation of the indicated proteins. (b) Confluent cells were exposed to either 21%, 1% or 0.1% O₂ for 14 h and then total and viable cells were counted. (c) Cells were exposed to 21% or 0.1% O₂ as in (a) and then either immediately treated with CA4P (1 μM) or vehicle for 15 min or returned to a normal oxygen incubator for 30 or 60 min to re-oxygenate (hypoxia-re-oxygenation, H/R) before they were treated with CA4P. (d) Cells maintained either in 21% or 0.1% O₂ for 14 h, were exposed to CA4P as in (a) and active GTP-bound RhoA was analysed in pull-down assay. (e) Active Rho was normalised to total RhoA and expressed as fold increase over control cells in 21% O₂. (f) pMLC data from (a) and (c) was normalised to actin and expressed as fold increase over control untreated cells maintained in 21% O₂. All data were analysed by one-way Anova followed by a Tukey post-test. Values are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). All blots and analyses are representative of 3–7 independent experiments.