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. 2021 Apr 13;12:2198. doi: 10.1038/s41467-021-22522-4

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© The Author(s) 2021

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 — a Quantification of the contractility of MCF10A WT (WT) and EFA6B KO55 (KO) cells transfected with a control siRNA, and EFA6B KO55 cells transfected with the indicated siRNA evaluated by a collagen gel contraction assay. Values are the mean surface area of the collagen gel ± SEM. N = 3, one-way ANOVA test with Dunnett’s multiple comparison p-values calculated versus KO55. b Expression of pMLC (phospho-myosin light chain) and total MLC (myosin light chain) analyzed by immunoblot in MCF10A WT and EFA6B KO55 cells, N = 4. c Representative images of the MCF10A WT and EFA6B KO55 spheroids embedded 2 days in collagen I stained for F-actin (red). The organization of the collagen I fibers surrounding the cell aggregates were imaged by confocal reflectance microscopy (green). The large inset is a 2× zoom-in image of the leader cell. Arrowheads point to thin membrane extensions co-localized with collagen fibers. Scale bars 20 μm. d Two days post-transfection of EFA6B KO55 cells with the indicated siRNAs, the expression of the corresponding protein was analyzed by immunoblot and quantified, N = 3. HSP60 and p85 served as loading controls. e Quantification of the percentage of aggregates (n = 100) with invasive protrusions of EFA6B KO55 cells grown in collagen I for 2 days after transfection with the indicated siRNAs. N = 4 for siCDC42 and N = 3 for siRHOA, siRHOC, siRAC, average ± SEM, one-way ANOVA test with Dunnett’s multiple comparison p-values. f Quantification of the percentage of invadopodia in EFA6B KO55 cells (n = 30) grown in collagen I for 2 days after transfection with CDC42 targeted siRNAs. N = 3, average ±SEM, one-way ANOVA test with Dunnett’s multiple comparison p-values. g Representative images of EFA6B KO55 cells transfected with the indicated siRNAs and stained for cortactin (green), F-actin (red), and nuclei (blue). Scale bars 20 μm. h Lysates of MCF10A WT and EFA6B KO55 cells were reacted with GST, GST-CRIB (CDC42GTP- and RAC1GTP-interacting domain of PAK) or GST-RBD (RHOAGTP- and RHOCGTP-binding domain of rhotekin) prebound to glutathione-sepharose beads. The whole lysates and bound proteins were analyzed by immunoblot with the indicated antibodies, N = 3. Source data are provided as a Source Data file.