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. 2018 Jul 5;8:10191. doi: 10.1038/s41598-018-28637-x

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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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Hypoxia-induced invadopodium precursors formation in MCF-7 cells requires CSRP2 upregulation. (A) Gelatin degradation assay showing that normoxic (upper panels) and hypoxic (lower panels) MCF-7 cells do not promote (Oregon Green 488-labelled) gelatin degradation after 48 h. After fixation, MCF-7 cells were stained for actin (in green) and cortactin (in red), and imaged using confocal microscopy. (B) Western blot analysis of the MT1-MMP and HIF-1α protein levels in normoxic and hypoxic MCF-7 cells. (C) MCF-7 cells were transfected with non-targeting or 2 different CSRP2 siRNAs (siCSRP2#1 and 2), cultured under normoxic or hypoxic conditions, and invadopodium precursors were detected by actin (green) and cortactin (red) co-labelling. (D) Quantitative analysis of invadopodium precursor density in MCF-7 cells as in (C). The data originate from 3 independent experiments (n = 60). (E) Gelatin zymography assay conducted with the conditioned media collected from MCF-7 cells cultured as in (C,D). The zymogram is representative of three independent experiments. Similar data were obtained with siCSRP2#2. (F,G) Hypoxic MCF-7 cells were labelled for actin (green), cortactin (red) and either N-WASP (immunolabelling, F) or Tks5 (Tks5-GFP, G). (H) CSRP2-GFP co-localizes with actin (red) at invadopodium precursors in hypoxic MCF-7 cells. The insets show magnification of invadopodium precursors. Bars = 15 µm; *p < 0.05; **p < 0.01.