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. 2022 Mar 1;12:842418. doi: 10.3389/fonc.2022.842418

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Copyright © 2022 Schneider, Winkler, Kell, Pfaffl, Atkinson and Moertl

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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Increased GRP78 expression in HNSCC EVs. (A, B) Nanoparticle tracking analysis of EVs collected via serial ultracentrifugation from cell culture supernatant of irradiated (6 Gy) and non-irradiated (0Gy) BHY cells. (C) Western Blot of BHY EVs with positive (Alix, TSG101, CD9) and negative (GAPDH, Calnexin) EV markers. (D, E) Western Blot analysis with representative blot (E) shows increased amounts of GRP78 in EVs derived from irradiated cells (6Gy-EVs) in comparison to EVs derived from non-irradiated HNSCC cells (0Gy-EVs). GRP78 expression was normalized to Ponceau S staining and 0 Gy served as baseline (n = 4, t-test, **p < 0.01). (F, G) Flow cytometry analysis of GRP78 surface expression on BHY EVs show increased GRP78 on 6Gy-EVs compared to 0Gy-EVs. CD63 was used as control surface antigen showing no significant change in radiation induced surface expression (n = 4, t-test, *p adj. < 0.05). (G) Example dot blots of bead-coupled EVs in flow cytometric analysis.