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. 2024 Jun 8;52(13):7665–7686. doi: 10.1093/nar/gkae487

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© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — KDM6A influence the genomic stability through regulating the stability and restart of DNA replication forks. (A) Copurified SND1 and DNA replication related proteins with KDM6A were validated by mass spectrometry and listed in table. (B) Interaction network of replication forks related proteins coimmunoprecipitated with KDM6A. (C) Detection of the chromatin associated KDM6A. Chromatin-associated proteins were prepared from the nucleus of HEK293T cells treated with either CPT or HU and were subjected to western blot. The number represents the quantification of KDM6A against to H2B. (D) Detection of KDM6A associated with nascent DNA in HeLa cells treated with CPT. Representative images are shown. Scale bars, 5 μm. (E) Western blot of RPA1 immunoprecipitation in HEK293T cells treated with HU for 1 h and followed by releasing into fresh media for indicated time. Copurified proteins were detected using specific antibodies. The number represents the quantification of KDM6A protein against to Flag-RPA1. (F) Western blot of nascent DNA-associated proteins at stalled replication forks. Nascent DNA-associated proteins were isolated in HEK293T cells treated with HU for 1 hour and followed by releasing into fresh media for indicated time using iPOND approach, and the resulted proteins were detected using specific indicated antibodies. (G) The replication forks foci labeled by EdU and BrdU in HeLa cells with HU. Representative images are shown. The histogram counted the number of EdU and BrdU foci in KDM6A-knockdown HeLa cells. Scale bars, 5 μm. * P < 0.05. P values were determined using a paired Student's t test. (H) The progression (left panel) and restart (Right panel) of replication forks were evaluated by DNA fiber assay. The histogram counted the length of DNA fiber labeled by CldU in KDM6A-deficient HeLa cells. (I, J) Detection of H3K9ac, H4K8ac, γH2AX (I) and Ku70 (J) associated with the nascent DNA in cells after KDM6A knockdown.