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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. 6 — STEF regulates myosin-generated tension on the nuclear envelope. a Atomic force microscopy measurement of nuclear stiffness (calculated as mean Young’s modulus) in Control and STEF KO MEFs (10 cells per condition). Representative replicate from two independent experiments. Statistical significance was verified using an unpaired t-test, * p < 0.05. b Schematic representation of the mini-Nesprin-2G tension sensor construct (mN2G-TS). c Representative spinning disc confocal images of control and STEF KO MEFs expressing the mN2G-TS construct, imaged using the TFP filter to determine construct localisation, and the FRET filter to calculate the FRET Index at the nuclear membrane. d Quantification of mN2G-TS FRET Index at the nuclear envelope in control MEFs, STEF KO MEFs, and control MEFs after treatment with a combination of 10 µM myosin light-chain kinase inhibitor (ML7) and 10 µM ROCK inhibitor (Y-27632). Values are presented as the mean of three independent experiments. Statistical significance was verified using an unpaired t-test, * p < 0.05, ** p < 0.01. e Representative confocal immunofluorescence images of control and STEF KO MEFs stained for DNA (Hoechst), pMLC and F-actin (Phalloidin). f Quantification of apical phospho-MLC positive cables over nuclear area from control and STEF KO MEFs (~20 cells per condition, per replicate). Values represent the mean of three independent experiments. Statistical significance was verified using a paired t-test. * p < 0.05. g qPCR for the TAZ target gene Ctgf, normalised to Tbp expression in control and STEF KO MEFs pre-treated with either DMSO or 10 µM ML7 for 2 h. Data are relative to the negative control (control MEFs + DMSO) and are presented as the mean from four independent replicates. Statistical significance was verified using a one-way ANOVA, using Tukey’s multiple comparison test to compare the means of each sample. *** p < 0.001, **** p < 0.0001. h qPCR for the TAZ target gene Ctgf, normalised to Tbp expression in liver tissue isolated from WT (n = 3) and STEF KO (n = 4) mice. Statistical significance was verified using an unpaired t-test, *** p < 0.001. All error bars represent S.E.M. Scale bars = 10 µm throughout