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. 2023 Sep 21;55(10):1721–1734. doi: 10.1038/s41588-023-01504-w

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© The Author(s) 2023

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 2 — a, Schematic of the A3B knock-out strategy resulting in an A3A/B fusion. CRISPR cleavage sites are indicated by arrows and the homologous gRNA-targeted region is shown below with PAM (red). Exons are indicated by colored boxes. b, Diagnostic PCR products distinguishing WT A3B and 29.9 kbp A3B deletion allele (**, clones not pertaining to this manuscript; sequence verified). c, Immunoblot of MCF10A WT and A3B KO derivative treated with DMSO or PMA (25 ng/ml, 24 hrs) and probed with the indicated antibodies (n = 3 independent experiments). d, DNA deaminase activity assay using extracts from MCF10A WT and A3B KO derivative treated with DMSO or PMA (25 ng/ml, 24 hrs; purified A3A positive control; reaction buffer negative control; n = 3 independent experiments). e, A3B gene schematic with an arrow indicating the exon 2 mRNA region targeted by an A3B-specific shRNA in depletion experiments (target sequence shown below). f, Immunoblot of U2OS shCtrl and shA3B cell lines probed with the indicated antibodies; (n = 3 independent experiments). g, DNA deaminase activity assay of extracts from U2OS shCtrl and shA3B cell lines (purified A3A was used as a positive control and reaction buffer as a negative control; n = 3 independent experiments). h, EdU staining of MCF10A WT and A3B KO cell lines (n = 1 with a minimum of 10,000 cells per condition). i, PI staining of MCF10A WT and A3B KO cell lines (n = 3 experiments with 10,000 cells per condition; mean ± SD). j, EdU staining of U2OS shCtrl and shA3B cell lines (n = 1 with 10,000 cells per condition). k, PI staining of U2OS shCtrl and shA3B cell lines (n = 3 experiments with 10,000 cells per condition; mean ± SD). l, A3B gene schematic with an arrow indicating the exon 3 gRNA targeting region (target sequence shown below). m, Immunoblot of whole cell extracts from U2OS WT and A3B KO cell lines probed with the indicated antibodies (n = 3 independent experiments). n, Immunoblot of whole cell extracts from U2OS WT and A3B KO cell lines transfected as shown and probed with the indicated antibodies (n = 2 independent experiments).

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