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. 2017 Jul 24;7:6219. doi: 10.1038/s41598-017-06676-0

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© The Author(s) 2017

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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Activated GR and NCoA-2 redistribute to colocalized intranuclear foci. Subcellular distribution of GFP-GR and mCherry-NCoA-2 in BHK cells was analyzed upon hormone induction by using different imaging tools as indicated in Methods section. (a) Cells expressing GFP fused to the indicated GR variant (wild type, GRdim, GRmon) were treated with Dex, 21-OH or vehicle (Veh). NCoA-2 was co-expressed where indicated (Scale bar: 5 µm). GR capability to form intranuclear foci was quantitatively analyzed by calculating the nuclear foci density (n = 15). Bars with the same superscript letters represent data not significantly different from each other (p < 0.05). (b) Subcellular distribution of GFP-GR and mCherry-NCoA-2 in untreated or Dex-stimulated cells (Scale bar: 5 µm). (c) Analysis of time-lapse images of cells co-expressing GFP-NCoA-2 and mCherry-GR, within 15 min after Dex-stimulation. Different image-analyisis tools were used to calculate the relative foci density (δ) (upper panel); percentage of colocalized foci (upper panel, yellow line) and the relative mean intensity of foci (lower panel). Light curves represent single cell data to illustrate the cell-to-cell variability while dark curves correspond to mean values. (d) Representative images of cells expressing mCherry-NCoA-2 and GFP fused to the indicated GR variant (wild type, GRdim, GRmon) and treated with Dex, 21-OH or Veh (Scale bar: 5 µm).