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. 2019 Sep 24;10:4333. doi: 10.1038/s41467-019-12275-6

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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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Fig. 2 — Characterization of endo-EVs derived from LNP-treated cells. a Nanoparticle tracking analysis for size distribution and concentration of EVs from untreated cells (Left side panel) and from MC3-LNP-treated cells (Right side panel). For each graph (one representative replicate), a table is provided, including mean and mode sizes, SD, and D-values (D10, D50, D90) and particle concentration. b EV-mRNA protection assay against RNase A. The hEPO-mRNA qPCR data is represented as scatter dot plot and mean SD of n = 3 biologically independent samples. c Cy5 mRNA in CD63/CD9 positive EVs. CD63⁺ EVs were stained against CD9 antibody and analyzed by FACS for Cy5 mRNA detection. The sole beads incubated with PBS instead of EVs are shown as negative control. Approximately 96% of immunoprecipitated EVs (50 µg assay) from untreated cells are positive for CD63 and CD9 but they are negative for mRNA. By contrast, ~88% of immunoprecipated EVs (50 µg assay) from LNP-treated cells are positive for CD63 and CD9, and 26% EVs contain mRNA that is secreted after the endocytosis of LNP-mRNA. The percentage of CD63/CD9 positive EVs containing Cy5 mRNA is presented in the upper right quadrant. The FACS dot plots represent Cy5 mRNA (y-axis) vs. CD9 (x-axis). One out of two biological replicates is shown. Source data are provided as a Source Data file