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. 2021 Mar 5;40:89. doi: 10.1186/s13046-021-01883-0

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 7 — EVs secreted by high-dose irradiated A549 cells induce in vitro abscopal effects. a EVs secreted by irradiated A549 stimulate the synthesis of DNA:RNA hybrid structures. Immunofluorescence staining with S9.6 antibody. The images are representative of unIR A549 cells exposed to EVs isolated from culture medium of A549 cells non irradiated or irradiated at different doses. Cell images were captured by Nikon Eclipse Ti2 confocal microscope with 60x plan apochromat oil immersion objective lens. Data are presented as grand median ± MAD. b EVs secreted by irradiated A549 induce senescent phenotype. (I) Representative images of SA-β-gal staining of UnIR A549 cells after exposure to EVs isolated from culture medium of IR A549 cells. II. Colony-forming assay (CFA). The cells were exposed to EVs isolated from UnIR or IR A549 cells for 14 days. 0.05% crystal violet solution was used to visualize the generated colonies with more than 50 cells, which were quantified under inverted microscope (Olympus IX51 microscope, Olympus Corporation, Tokyo, Japan) by two independent observers. III. Expression levels of senescence markers. p21, IL6 mRNA levels were measured by Real-Time PCR and normalized to GAPDH and HPRT-1. Data are the mean of two independent experiments. (C) EVs secreted by irradiated A549 induce M1 polarization in a murine macrophage cell line. Total RNA from RAW 264.7 M0 cells (see Methods section) exposed to EVs secreted by unirradiated, 10 Gy or 20 Gy-irradiated A549 cells were analyzed by RT-PCR for the expression of representative murine M2 genes (Arg1, Egr2) and M1/pro-inflammatory cytokines (IL-6, IL-1β). Expression data are given as fold increase over the mRNA level expressed by RAW 264.7 M0 exposed to UnIR EVs. Data are represented as mean ± SD of triplicate values of two independent experiments (*p < 0.05, **p < 0 .01 ***p < 0.001)