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. 2019 Sep 3;55(10):838–853. doi: 10.1007/s11626-019-00403-x

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© The Author(s) 2019

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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.

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Figure 4. — YKL-40 expression in D492HER2 is linked to increased potential to stimulate angiogenesis. (a) Conditioned media (CM) from D492HER2 cells stimulates angiogenesis in an in vitro angiogenesis assay. This effect was abrogated upon YKL-40 neutralization by a monoclonal antibody (mA^YKL40) but rescued by the addition of recombinant YKL-40 protein (YKL40^r) (Scalebar = 200 μm). (i) Parameters of angiogenesis were measured and analyzed by ImageJ angiogenesis analyzer plug (mean ± SD) and significance was assessed with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (*p ≤ 0.05). (ii) Decreased concentration of YKL-40 upon incubation of cells with mA^YKL40 was verified with ELISA. Significance was tested with an unpaired t-test (****p ≤ 0.0001). (b) When YKL-40 was overexpressed in D492 and D492M, there was an increase in the capillary network formation, whereas stable knockdown of YKL-40 in D492HER2 showed a tendency to reduced capillary network formation. (c) Knockdown of YKL-40 reduced the expression of other pro-angiogenic inducers, VEGFA and VEGFC. (d) Finally, recombinant YKL-40 protein (YKL-40^r) induced proliferation of endothelial cells when added to the media. Unpaired t test was used to test significance (**p ≤ 0.01, ***p ≤ 0.001).