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. 2023 Feb 9;14:706. doi: 10.1038/s41467-023-35992-5

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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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Fig. 1 — a Workflow for establishing an HCT116-POLD1-AID2 cell line expressing only POLD1 that is tagged with OsTIR1(F74G) (mAC). b Experimental workflow for assessing POLD1 protein degradation following different periods of incubation with 5-Ph-IAA. c Western blot (WB) analysis of POLD1-mAc in asynchronous cells treated as shown in panel b. β-tubulin was a loading control. d Experimental workflow for analysis of MiDAS in prometaphase HCT116-POLD1-AID2 cells following APH treatment, enrichment of G2 cells by RO3306, and POLD1-mAC degradation. POLD1-mAC depletion was induced by treating cells with 5-Ph-IAA for 2 h. e WB analysis of POLD1-mAC (detected with a POLD1 antibody) following 2 h 5-Ph-IAA treatment. β-tubulin was used as a loading control. Representative images (f) and quantification (g) of MiDAS foci (labeled with EdU; red) in prometaphase cells treated as shown in panel d. h Experimental workflow for U2OS cell synchronization and analysis of MiDAS in prometaphase cells following REV3 or REV1 depletion. In b and h, before being incubated with EdU for 30 min (with or without 5-Ph-IAA), cells were rinsed three times with pre-warmed, drug-free culture medium within 5 min. i Top, WB analysis of REV3FLAG following transfection of a REV3FLAG expressing plasmid together with control or REV3 siRNAs. Flag antibody was used to detect flag-REV3, since a reliable REV3 antibody is not available. Actin was used as a loading control. Protein sample from untransfected U2OS cells was used as a negative control. Lower, WB analysis of REV1 after transfecting cells with control or REV1 siRNAs. β-tubulin was used as a loading control. Representative images (j) and quantification (k) of MiDAS foci (labeled with EdU; red) in prometaphase cells treated as shown in panel h. DNA was stained with DAPI (blue). Each data point in charts of g and k is means of three independent experiments and plotted with Prism (n = number of cells analyzed in each condition in three independent experiments). Error bars represent SEM. P values were calculated using a two-tailed non-parametric Mann–Whitney test. Scale bars, 10 μm. Hr hour, min minute.