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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. 6 — A549 cells grown as 3D models confirm the irradiation-induced SASP characterized by high synthesis of DNA:RNA hybrids structures. a Representative images of SA-β-gal staining performed on FFPE tissue sections of A549 spheroids fixed after exposure to 10 or 20 Gy. For evaluation of cells positive to SA-β-Gal assay, a homemade Matlab tool was used. The appropriated mask needed to identify the cells positive to SA-β-Gal assay in the investigated area was obtained using the function imageSegmenter; (* p < 0.05; *** p < 0.0001) (b) mRNA expression of SASP biomarkers detected in A549 cells grown as 3D spheroids using GAPDH and HPRT as housekeeping genes. Data are presented as mean ± SD. c, d Confocal immunofluorescence images representative of A549 spheroids after different irradiation doses showing cytoplasmic (green) and nuclear (white) localization of DNA:RNA hybrids (scale bar 10 μm). The micrograph shows the co-localization analysis performed through confocal high-resolution analysis. Bar graphs, mean ± error bars of fluorescent signals in the different compartments are also reported (see Methods section) (* p < 0.05)