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. 2018 Feb 5;9:493. doi: 10.1038/s41467-017-02640-8

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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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Fig. 2 — Nuclear dynamics of NF-κB and IRF3: pulse-like after LPS, switch-like binary after poly(I:C). Nuclear translocation of a transcription factor is defined here throughout as a normalised quantification of its nuclear fluorescence in confocal images. RelA and IRF3 use a normalisation relating nuclear to whole-cell fluorescence, which underlies the histograms, fractions of responding cells and scatter plots. Distinct normalisations were used for p-STAT1 (to account for its changing cellular level) and for trajectories in RelA-GFP MEFs (see Methods). a, b Time course of nuclear localisation of RelA (NF-κB), IRF3, IκBα and p-STAT1 in response to poly(I:C). Scale bars: 50 μm. WT MEFs were stimulated with 1 μg/ml poly(I:C), fixed and stained at given time-points with antibodies against RelA and IRF3, RelA and IκBα, or p-STAT1. a Histograms show the full time course. Representative excerpts from confocal images show cells after 0 (nt), 4 and 10 h of poly(I:C) stimulation. b Changes in fraction of cells responding to poly(I:C) in time. A cell was deemed responding if its nuclear translocation exceeded a threshold based on nuclear fluorescence of non-treated cells. c Nuclear RelA trajectories in RelA-GFP MEFs stimulated with 1 μg/ml or 3 μg/ml poly(I:C) for 24 h. Sample time-lapse confocal microscopy images showing nuclear translocation of GFP-tagged RelA in response to poly(I:C) are provided in Supplementary Movies 1 and 2. d Time course of nuclear localisation of RelA and IRF3 in response to LPS. WT MEFs were stimulated with 1 μg/ml LPS, fixed and stained at given time-points with antibodies against RelA and IRF3. Histograms show the full time course. Representative confocal images show cells after 0 (nt), 1 and 4 h of LPS stimulation. e Nuclear RelA trajectories in RelA-GFP MEFs stimulated with 1 μg/ml LPS for 10 h. Sample time-lapse confocal microscopy images showing nuclear translocation of GFP-tagged RelA in response to poly(I:C) are provided in Supplementary Movie 3. Histograms (n ≥ 500) show a representative experiment out of 3. See Supplementary Data 1 and 2 for corresponding uncropped immunostaining images