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. 2019 Oct 7;9:14363. doi: 10.1038/s41598-019-50915-5

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© The Author(s) 2019

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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Gelatin degradation assay. (a,b) Representative fluorescent images of microscopic fields where HUVEC cells transfected with control-siRNA (a) or Tks4-siRNA (b) and seeded at the same cell density digested fluorescent OregonGreen488-conjugated gelatin substrate for 20 hours. Dark areas correspond to cleaved substrate. Scale bar: 100 μm. (c) Quantitative analysis of digested areas from n = 3 independent sets of experiments. Error bars are SEM, asterisk (*) indicates statistically significant difference with Student’s t-test, $p < 0.05$ . (d,e) HUVEC cells transfected with control-siRNA (d) or Tks4-siRNA (e) and seeded on fluorescent OregonGreen488-conjugated gelatin substrate (green) for 5 hours were immunolabeled with anti-cortactin antibody (blue) and stained with phalloidin-TRITC to visualize F-actin (red). Arrowheads in d point to cortactin positive podosome structures. For better identification of areas with increased proteolytic activity, insets in d and e show the corresponding green fluorescent channel only. Scale bars: 10 μm.