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This in turn, leads to DNA damage\u2014which accumulates in specific genomic regions\u2014contributing to genomic instability in cancer. However, the interplay between oncogene-induced DNA damage and chromatin organization is still poorly understood. In this study, we introduce a QUantitative ANalysis of DNA cOunterstains (QUANDO) to investigate the subnuclear localization of DNA damage in single-cell nuclei of U937-PR9 cells, an in vitro model of acute promyelocytic leukemia (APL). Using advanced imaging techniques, including DNA intensity analysis and colocalization by image cross-correlation spectroscopy (ICCS), we map DNA damage foci and correlate them with chromatin regions of different density. QUANDO is applied to dual-color confocal images of the DNA damage marker \u03b3-H2AX and the DNA counterstain DAPI, allowing single-cell measurements of foci distribution within areas of low or high DNA density. We find that spontaneous DNA damage and DNA damage induced by the activation of PML-RAR\u03b1 oncogene predominantly localize in euchromatic regions. Conversely, when DNA damage is induced by the radiomimetic agent neocarzinostatin (NCS), the foci appear more evenly distributed in euchromatic and heterochromatic regions. These findings underscore the complex interplay between oncogene activation and chromatin organization, revealing how disruptions in DNA damage distribution can contribute to genomic instability and offering new insights for targeting DNA repair mechanisms in cancer therapies.<\/jats:p>","DOI":"10.1007\/s00249-025-01755-x","type":"journal-article","created":{"date-parts":[[2025,5,7]],"date-time":"2025-05-07T02:40:53Z","timestamp":1746585653000},"page":"165-173","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Location of oncogene-induced DNA damage sites revealed by quantitative analysis of a DNA counterstain"],"prefix":"10.1007","volume":"55","author":[{"given":"Greta","family":"Patern\u00f2","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Silvia","family":"Scalisi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gaetano Ivan","family":"Dellino","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mario","family":"Faretta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pier Giuseppe","family":"Pelicci","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alberto","family":"Diaspro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6539-394X","authenticated-orcid":false,"given":"Luca","family":"Lanzan\u00f2","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,5,7]]},"reference":[{"key":"1755_CR1","doi-asserted-by":"publisher","first-page":"1096","DOI":"10.1038\/s42003-023-05468-9","volume":"6","author":"H Balasubramanian","year":"2023","unstructured":"Balasubramanian H, Hobson CM, Chew T-L, Aaron JS (2023) Imagining the future of optical microscopy: everything, everywhere, all at once. 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