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. 2020 Jan 10;7:368. doi: 10.3389/fcell.2019.00368

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Copyright © 2020 Li, Chen, Kemper and Brigstock.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Characterization and actions of EVs from HepG2 cells. (A) EVs from 48-hr HepG2 cell conditioned medium (serum-free) were analyzed by NTA or Western blotting (inset, with comparison to HepG2 cell lysate; 10 μg EV or cellular protein per lane). (B) P3-4 mouse HSC were incubated for 42 h in the absence or presence of HepG2 cell-derived EVs (8 μg/ml) and then analyzed by RT-PCR for expression of CCN2, αSMA, or Col1a1. (C) PKH26-labeled EVs underwent iodixanol gradient centrifugation and (D) the peak fluorescent fraction was tested on mouse HSC as in panel (B). (E) AlamarBlue viability assay or (F) CyQuant proliferation assay of HepG2 cells that were treated with 20 mM CCl₄ (or 0.02% DMSO carrier) for 24 h followed by 8 or 16 μg/ml HepG2 cell-derived EVs for the following 24 h. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.005.