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. 2018 May 8;8:7139. doi: 10.1038/s41598-018-25461-1

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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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Fibroblasts increase cell number by increasing viability and reducing apoptosis. Cultures were dosed with a vehicle control or 100 nM E2 for 72 hours. (A) Total number of nuclei was measured to evaluate cell number. (B) Proliferation was measured by the percentage of cells positive for proliferation marker Ki67. (C) A live/dead assay was used to evaluate cell viability, where the cells were stained with calcein, Hoescht, and ethidium homodimer. (D) Cell apoptosis was measured by quantifying the number of nuclei positive for activated caspase-7, which is a key protein in the apoptosis cascade. A two-way ANOVA was run to evaluate interactions between conditions, followed by a Tukey’s multiple comparisons test to identify significant differences. *= vs. respective vehicle (p < 0.05) and ‡= vs. respective monoculture (p < 0.05). Graphs represent data from three independent experiments.