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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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GR and NCoA-2 dynamically partition between the nucleoplasm and DNA-dependent discrete foci. Properties of GR nuclear foci in BHK cells were analyzed using biochemical and FCS-based approaches. (a) Cells co-expressing GFP-GR and H2B-mCherry were fixed, permeabilized and digested with RNase A or DNase I (Scale bar: 5 µm). (b) In situ-DNase I digestion of cells co-expressing GFP-GR and mCherry-NCoA-2 before and after stimulation with Dex (Scale bar: 5 µm). (c,d) Orbit-scanning FCS experiments were run in cells expressing either GFP-GR or GFP-NCoA-2 and mCherry-GR. (c) The laser orbits were set to scan through foci which position were further determined with subpixel precision. The intensity traces of foci were then used to calculate the autocorrelation or cross-correlation functions (representative curves). ACF data was fitted with a model that considers binding to fixed targets (continuous lines). (d) Mean residence time of mCherry-GR (dark red), GFP-NCoA-2 (green) and CCF (yellow) at foci (n = 12). Red bar corresponds to the residence time of GFP-GR in cells that were not cotransfected with NCoA-2. Bars with the same superscript letters represent data not significantly different from each other (p < 0.05).