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. 2018 May 29;9:2124. doi: 10.1038/s41467-018-04404-4

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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. 4 — Rac1 activity at the nuclear envelope regulates the actin cap. a Representative spinning disc confocal images of live parental U2OS and STEF-depleted CRISPR clones (CRISPR#1 and CRISPR #2) expressing the Raichu-Rac1 FRET probe, showing YFP signal intensity, FRET intensity as calculated from CFP/YFP and the FRET intensity in an isolated region of interest (ROI) around the nuclear envelope. Scale bar = 10 µm. b Quantification of the FRET ratio in the perinuclear region of parental U2OS and STEF-depleted CRISPR clones (CRISPR #1 and CRISPR #2). Data shown are pooled from three independent replicates. Statistical significance was verified using a one-way ANOVA, using Dunnett’s multiple comparison test to compare the means of the CRISPR #1 and CRISPR #2 to the control. **** p < 0.0001. c Schematic representation of the structure of the eGFP-Rac1-KASH-Ext construct and its localisation at the nuclear envelope. d Representative confocal images of control MEFs expressing a GFP-only vector (top row) and STEF KO MEFs expressing either a GFP-only vector (second row), nuclear envelope localised constitutively active Rac1, eGFP-V12-Rac1-KASHext (third row), or nuclear envelope localised WT Rac1, eGFP-WT-Rac1-KASHext (fourth row). All MEFs are stained for DNA (Hoechst), GFP and F-actin (Phalloidin). Scale bar = 10 µm. e Quantification of apical actin cable number over nuclear area from cells as in d. Values represent the mean of three independent experiments ( >10 cells per condition, per replicate). Statistical significance was verified using a one-way ANOVA, using an uncorrected Fisher’s LSD multiple comparison test to compare the means of each sample. * p < 0.05, n.s. = not significant. f Representative widefield immunofluorescence images of control MEFs either untransfected (top row) or expressing nuclear envelope localised dominant negative Rac1, eGFP-N17-Rac1-KASHext (bottom row), stained for DNA (Hoechst), GFP and F-actin (Phalloidin). Scale bar = 5 µm. g Quantification of apical actin cable number over nuclear area from cells as in f. Values represent the mean of three independent experiments ( > 15 cells per condition, per replicate). Statistical significance was verified using a paired t-test. * p < 0.05. Error bars represent S.E.M. throughout