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. 2019 Dec 9;77(20):4093–4115. doi: 10.1007/s00018-019-03399-5

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© The Author(s) 2019

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Characterization of EVs secreted by MV3 human metastatic melanoma cancer cells with stable, inducible expression of GFP-HAS3. a 3D projection of live GFP-HAS3 expressing cell grown in 3D collagen gel. Confocal sections of CD44-immunostained uninduced (b) and induced (c) MV3 cells and a higher magnification image of an area indicated by a white box in (c) is shown in (d). Panels e–h show HA staining of EV preparations isolated from GFP-HAS3 expressing cells, and h shows a higher magnification image of the area indicated by a black box in (g). i Western blotting of GFP-HAS3 detected with GFP antibody and other typical EV markers. Nanoparticle tracking analysis of EV levels (j) and size distribution (k) secreted from uninduced (MV3-EVs) and induced (HAS3-EVs) MV3 cells, and l transmission electron microscopy of EVs isolated from GFP-HAS3 expressing cells. Data represent mean ± S.E. from three independent experiments; *P < 0.05 (one-way ANOVA, Tukey’s test). Arrows in all panels indicate EVs. Scale bars represent 20 µm in (c), 2 µm in (d) and (h), 10 µm in (g) and 200 nm in (j)