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. 2018 Sep 21;9:3846. doi: 10.1038/s41467-018-06368-x

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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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Fig. 6 — Cx43 carboxy-terminal controls N-cadherin by localizing into the nucleus. a Hela cells transfected with GFP-tagged Cx43 forms as indicated (in green), red: phalloidin, blue: DAPI. Scale bar = 25 μm. b–i Inducible construct to control nuclear localization of Cx43-20k in neural crest and Hela cells. b Diagram of Cx43-20k-GR-GFP function. GR: glucocorticoid receptor domain. When Cx43-20kGR is expressed in a cell, it binds to HSP90 which sequestrates it into the cytoplasm; upon addition of the ligand (dex: dexamethasone) Cx43-20kGR is released and it can go into the nucleus. c St23 neural crest expressing Cx43-20kGR-GFP with (+) or without (−) dxm. Note that GFP fluorescence is uniformly distributed in -dxm cells, but it becomes localized in the nucleus after dxm treatment. Chart in c shows Cx43-20kGR nuclear fluorescence normalized to the cytosolic fluorescence (n_−dxm = 78, n_+dxm = 89 cells, N = 3). Scale bar = 20 μm. d qPCR for n-cad of st20 embryos (N_{from bars1–4} = 4, 4, 5, 5). e Activation of Cx43-20k nuclear localization by dxm rescues n-cad levels, analyzed by ISH for twist in embryos at st24. White arrow shows injected side and green line the neural crest. f % of n-cad expressing embryos shown in e (n_CMO = 83, n_CxMO = 95, n_−dxm = 67, n_+dxm = 64, N = 4). g St24 embryos showing neural crest migration by ISH of twist; arrowheads: normal migration, brackets: impaired migration, asterisk: eye. Scale bars in e, i, and g = 50 μm. h % of embryos with normal neural crest migration analyzed by ISH for twist; embryos are shown in g (n_CMO = 91, n_CxMO = 102, n_−dxm = 87, n_+dxm = 93, N = 5). i HeLa cells were transfected with EGFP or with Cx43-20kGR-GFP and treated with DMSO or dxm. Chart in i shows Cx43-20kGR-GFP nuclear fluorescence normalized to the cytosolic fluorescence (n_−dxm = 56, n_+dxm = 72 cells, N = 3); Scale bar = 25 μm. Histograms in c, d, f, h, and i represent mean ± S.E. (one-way ANOVA p < 0.001, two-tailed t test p^** < 0.01, p^*** < 0.001). N number of independent experiments; n sample size. Spread of data in bar charts is shown as overlying dots. n.s. nonsignificant