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. 2018 Jul 19;8:10909. doi: 10.1038/s41598-018-29101-6

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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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NMac1 induces morphological and other changes in MDA-MB-231 cells. (a) NMac1 reduces membrane ruffle in MDA-MB-231 cells. Localization of F-actin was analyzed by confocal microscopy in control and NMac1 treated cells. MDA-MB-231 cells treated with NMac1 25 µM (or 0.05% DMSO as vehicle) for 16 h were stained by phalloidin-Rhodamine. Number of cell ruffles of MDA-MB-231 cells was quantified by determining the ruffling index. (b) NMac1 reduces Rac1 activation. Active Rac1 pulldown assay was conducted in control and NMac1 treated MDA-MB-231 cells with indicated concentrations for 16 h. A representative experiment out of 3 independent experiments is shown. (c) NMac1 reduces Rac1 activation via Nm23-H1. Active Rac1 pulldown assay was conducted in control and NMac1 treated Nm23-H1 or (/and) H2 knocked down MDA-MB-231 cells with indicated concentrations for 16 h. A representative experiment out of 3 independent experiments is shown. (d) NMac1 reduces membrane ruffle in MDA-MB-231 cell via Nm23-H1. Localization of F-actin was analyzed by confocal microscopy in control and NMac1 treated Nm23-H1 or/and H2 knocking down MDA-MB-231 cells. At 48 hours after Nm23-H1 or/and H2 knocking down, MDA-MB-231 cells were treated with NMac1 25 µM (or 0.05% DMSO) for 16 hours followed by staining with phalloidin-Rhodamine.