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Fig. 8 | Journal of Experimental & Clinical Cancer Research

Fig. 8

From: TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer

Fig. 8

Correlation between DNA:RNA hybrids and retrotransposon ORF1. a Immunofluorescence staining with ORF1 antibody. The images are representative of A549, A549sh/p53 cells exposed to 10 or 20 Gy. Scale bar is 50 μm. Data are presented as grand median ± MAD. b DNA:RNA ORF1 co-localization analysis. Images are representative of A549 cells exposed to 10 Gy or 20 Gy. Scale bar is 50 μm. The average values of Mander’s ovelap (% of pixels that are overlapped, ± SEM) were calculated by software NIS-Element software’s tool (Nikon) and at least 20 cells were evaluated for each condition (*p < 0.05, **p < 0.01). c ORF1 and ORF2 mRNA expression in A549 and A549sh/p53 (see Methods section). Data are represented as mean ± SD of triplicate values of two independent experiments. d Representative confocal micrographs showing the positivity of A549 or A549sh/p53 cells to S9.6 antibody after exposure to Efavirenz (EFVZ) 20 μM, 10 Gy alone or followed by EFVZ 20 μM. Scale bar is 10 μm. (*p < 0.05, **p < 0.01 ***p < 0 .001). e Colony-forming assay (CFA). UnIR A549 cells were exposed to EVs isolated from unirradiated, irradiated, efavirenz-treated, and efavirenz-treated and irradiated A549 cells. 0.05% crystal violet solution was used to visualize the generated colonies with more than 50 cells, which were quantified under inverted microscope (Olympus) by two independent observers. f Dot blot analysis of RNA extracted from exosome or microvesicle fraction 72 h post-irradiation and immunoblotted with S9.6 antibody. The EVs and MVs were isolated from A549 cells exposed to different treatments. g Diagram illustrating the p53-mediated abscopal effect induced by radiotherapy. (I) Radiotherapy induces cell damage that leads to cell cycle arrest, necrosis, programmed cell death and cellular senescence. In particular, the cells bearing functional p53 may acquire SASP and release cytokines, inflammatory- (DAMPs) or senescence- (SAMPs) associated molecules. In addition, radiotherapy in the presence of wtp53 activates retrotransposon elements which, in turn, increase the level of genotoxic stress. SASP cells also release EVs conveying DNA:RNA hybrids and /or retrotransposon which, outside the field of irradiation (II), activate autodestruction mechanisms such as cellular senescence, apoptosis and innate immunity in p53-competent tumor cells

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