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. 2023 Jan 6;9:2. doi: 10.1038/s41421-022-00510-2

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© The Author(s) 2023

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. 1 — a SEM images of 2-E-transfected Vero E6 cells and EVs isolated from the transfected cells. Scale bar, 5 μm. b The flow chart showing the isolation of 2-E-EVs. c Diameter variation of 2-E-EVs. d Microscopy images of 2-E-mCherry-expressing Vero E6 cells during cell death. Five stages of 2-E-mCherry-transfected cells are shown. White arrows indicate 2-E-EVs. Scale bar, 10 μm. e TEM images of 2-E-transfected Vero E6 cells and 2-E-EVs. Yellow triangles, SMVs; red box, swelling Golgi; red arrows, damaged mitochondria; yellow dotted circles, lysosomes. Scale bars, 2 μm (up), 0.2 μm (bottom). f Microscopy images of Vero E6 cells. The cells were infected with a lentivirus vector (Lenti-mNeonGreen-3×Flag) and then transfected with 2-E-mCherry plasmid to induce vesicles. Scale bar, 10 μm. Illustration in the left corner shows a schematic view of the virus and plasmids used. An mCherry (red) and an mNeonGreen (green) tag were added to the C-termini of 2-E and lentivirus, respectively. g Left: immunoblot showing mCherry and Flag signals in isolated 2-E-EVs; all lanes were loaded with the same amount of total proteins. Right: nucleic acid gel image showing lentivirus in isolated 2-E-EVs. Shown is one representative experiment of three. h Schematic representation of infection experiments. Vero E6 cells were first transfected with the 2-E-mCherry plasmid and then infected with SARS-CoV-2. The next day, the cells, supernatant and 2-E-EVs were collected. i qRT-PCR analysis of SARS-CoV-2 copies after transfection with the 2-E-mCherry plasmid and infection. The histograms show viral copies in cells (left), supernatant (middle), and isolated 2-E-EVs (right). Data are means ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t-test.