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. 2023 Mar 23;14:1602. doi: 10.1038/s41467-023-37021-x

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© The Author(s) 2023

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. 3 — a, b IP analyses of nesprin-2 protein complexes in Met4 cells, Mena-depleted Met4 cells (Met4 Mena^−/−) and Met4 Mena^−/− cells with Mena11a re-expressed (Met4 Mena^−/− +Mena11a). Actin (a) and lamin A/C (b) were detected by western blotting; respective densitometric intensities were normalised (norm.) to nesprin-2 and expressed relative (rel.) to Met4 cells (right panels). Black bar, median; light grey box, range. Statistical analysis, Welch’s one-way ANOVA with two-stage Benjamini–Krieger–Yekutieli correction (n = 6 independent experiments) for (a), one-way ANOVA with Tukey’s correction (n = 5 independent experiments) for (b). c Spinning-disk confocal imaging of Met4 cells and Met4 Mena^−/− cells in 3D collagen matrix. Orthogonal projections (xz) were extracted from 3D brightest-point projections. Nuclei were detected using DAPI; F-actin was detected using phalloidin. Inverted lookup tables were applied. Images are representative of two independent experiments. Scale bar, 20 μm. d Quantification of nuclear volume of cells in 3D matrix (see c). 3D volume renderings of exemplar nuclei detected using DAPI are displayed. Black bar, median; dark grey box, 95% confidence interval; light grey silhouette, probability density. Statistical analysis, two-sided Welch’s t-test (n = 10 cells from n = 2 independent experiments). e Spinning-disk confocal imaging of Met4 cells, Met4 Mena^−/− cells and Met4 Mena^−/− +GFP-Mena11a cells on 2D fibronectin matrix. Orthogonal projections were extracted from 3D brightest-point projections. Nuclei were detected using DAPI. Inverted lookup tables were applied. Images are representative of four independent experiments. Scale bar, 20 μm. f Quantification of nuclear height of cells on 2D matrix (see e). 3D volume renderings of exemplar nuclei detected using DAPI are displayed. Black bar, median; dark grey box, 95% confidence interval; light grey silhouette, probability density. Statistical analysis, Kruskal–Wallis test with Dunn’s correction (n = 92, 87 and 94 cells for Met4, Met4 Mena^−/− and Met4 Mena^−/− +GFP-Mena11a cells, respectively, from n = 4 independent experiments). n.s. not significant.