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. 2024 Oct 5;15:8648. doi: 10.1038/s41467-024-52916-z

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 3 — a U2OS (EGFP-HR/STGC) WT or 53BP1-KO cells stably expressing shRNAs targeting PIF1 or POLD3 were infected with lentiviruses encoding I-SceI, and the percentage of EGFP-positive cells was analyzed by FACS, 5 days post-infection. The expression of PIF1 and POLD3 was examined by qPCR (Supplementary Fig. 3a). (n = 5 replicates). b BIR frequency in U2OS (EGFP-BIR/LTGC) WT, BRCA1-ΔBRCT and 53BP1-KO/BRCA1-ΔBRCT cells (left), and HR frequency in U2OS (EGFP-HR/STGC) WT cells expressing vector, or in BRCA1-ΔBRCT cells expressing 53BP1 shRNA or vector (right), were determined by FACS analysis of EGFP positive cells, 5 days after I-SceI lentiviral infection. (n = 5 replicates). c. U2O (EGFP-HR/STGC) WT and BRCA1-ΔBRCT cells were infected with lentiviruses expressing 53BP1 shRNA or vector, followed by a second round of lentiviral infection with shRNAs targeting PIF1 and POLD3 using vector as a control. HR frequency was determined by FACS analysis of EGFP-positive cells, 5 days after I-SceI lentiviral infection. The expression of indicated proteins was examined by qPCR (Supplementary Fig. 3c). (n = 5 replicates). d BIR frequency was determined in U2OS (EGFP-BIR/LTGC) WT and 53BP1-KO cells by FACS analysis 5 days after lentiviral infection of gRNA/Cas9^WT (left) or gRNA/Cas9^D10A (right). (n = 5 replicates). e U2OS (EGFP-BIR/LTGC-Flex1) cells expressing 53BP1 shRNA with vector as a control were infected with lentiviruses encoding FANCM shRNA or vector (left), or synchronized to S-phase using double thymidine block followed by HU treatment (2 mM, 24 h, right). BIR frequency was determined by FACS analysis of EGFP positive cells 6 days after. The expression of FANCM was examined by qPCR (Supplementary Fig. 3d). (n = 5 replicates). f BIR frequency was determined by FACS analysis of EGFP positive cells in U2OS (EGFP-BIR/LTGC) WT, 53BP1-KO, BRCA1-ΔBRCT and 53BP1-KO/BRCA1-ΔBRCT cell lines, 5 days after lentiviral infection of gRNA/Cas9^WT (left) or gRNA/Cas9^D10A (right). (n = 5 replicates). Source data are provided as a Source data file.