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. 2018 Jul 30;9:2980. doi: 10.1038/s41467-018-05367-2

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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. 2 — ER-mediated actin cytoskeletal remodeling induces suppressive cortical actin bundles (SCABs). a Leading edge kymography in representative time-lapse movies (Supplementary Movie 1) of control and E2-treated MCF7 cells (cultured under hormone starvation conditions), and control and ER-inhibited cells (cultured in regular media). Left panels indicate position at which kymographs were registered (line), and middle panels show minimum intensity projections from entire time series (Min. Proj.); scale bar is 10 µm. Right panels show corresponding kymographs; vertical scale bar is 10 µm; horizontal scale bar is 5 min. b Membrane ruffle quantification; mean ± s.d; *p = 0.03, ***p = 0.0005, ****p < 0.0001 (Welch’s t test). c Ruffling speed quantification; mean ± s.e.m; *p = 0.01, ***p = 0.0007, ****p < 0.0001 (Welch’s t test). Data from three independent experiments (n ≥ 45 cells per group). d Leading edge kymography in representative time-lapse movies (Supplementary Movie 2) of T47D cells. e Membrane ruffle quantification; mean ± s.d; *p = 0.03, ***p = 0.0002, ****p < 0.0001 (Welch’s t test). f Ruffling speed quantification; mean ± s.e.m; *p = 0.01, **p = 0.004, ****p < 0.0001 (Welch’s t test). Data from two independent experiments (n ≥ 30 cells per group). g Volumetric analysis of leading edge actin. Illustrative confocal z-series and corresponding 3D binary mask, demarcated as the area within 3 µm of cell edge and used to quantify leading edge fluorescence intensity. h Maximum intensity projections of z-series of E2-treated or fulv-treated MCF7 cells (Supplementary Movie 4), and 3D reconstructions of boxed areas; F-actin (red), Arp2/3 (green), and pMLC (magenta). Scale bar is 10 µm. i Quantification of leading edge Arp2/3 and pMLC in MCF7 cells. Data from three independent experiments (n = 21 cells per group; mean ± s.e.m). By two-way ANOVA, interaction analysis shows significant differences at p < 0.0001 (df = 3); pMLC values are significantly different in E2-treated group compared to other groups (p < 0.001). Arp2/3 values are not significantly different between groups. j Maximum intensity projections of z-series of E2-treated or tam-treated T47D cells and 3D reconstructions of boxed areas. F-actin is red, Arp2/3 in green, and pMLC in magenta. Scale bar is 10 µm. k Quantification of leading edge Arp2/3 and pMLC in T47D cells. Data from three independent experiments (n = 21 cells per group; mean ± s.e.m). By two-way ANOVA, interaction analysis shows significant differences at p < 0.0001 (df = 3); pMLC values are significantly different in E2-treated group compared to other groups (p < 0.001). Arp2/3 values are not significantly different between groups