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. 2019 Jul 19;9:10522. doi: 10.1038/s41598-019-47023-9

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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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EVs uptake assay. (A) DSP2-expressing target cells were preloaded with Enduren and incubated with DSP1-CD9 or DSP1-CD9/DSP2 EVs to be measured either at 6 or 24 h. Alternatively, DSP2-expressing target cells were incubated with EVs for 24 h, lysed and measured after Stop&Glo Renilla luciferase substrate addition. Signal of isolated DSP1-CD9 or DSP1-CD9/DSP2 EVs is also shown. Statistics: Statistics: ****. Two way Anova, Tukey’s multiple comparison test. n = 4. (B) DSP1-CD9/DSP2 EVs were incubated with supernatants or with mechanical obtained lysates from SUM159 WT cells preloaded with Enduren during 2 or 6 hours. EVs also were added to alive cells preloaded with Enduren at 2 or 6 hours. Controls of EVs incubated with Enduren or Stop&Glo reagent without EVs lysis are also shown. Luminescence signal from EVs with the different reagents and conditions at shown time points of incubation are depicted. Statistics: *** comparisons between EVs in the presence of cell lysates or alive cells at 0.5 hours; ### compared to EVs in the presence of cell supernatants and controls. Two way Anova, Tukey’s multiple comparison test. n = 3 (C) EV uptake kinetics using DSP2 or untransduced (WT) as target cells, pre-loaded with Enduren and incubated with DSP1-CD9/DSP2 EVs. Signal of either cells or EVs alone are also depicted. Statistics: **** compared to negative controls. Two way Anova, Tukey’s multiple comparison test. n = 4.