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. 2023 Jul 24;6:771. doi: 10.1038/s42003-023-05148-8

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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. 3 — a Immunofuorescence analysis of DPPA3 expression in WT, Zfp296-KO #98, and Rescue #22 ESCs. Nuclei were counterstained with DAPI. Scale bars, 50 μm (upper panels), 25 μm (middle and lower panels). b Western blot analysis of DPPA3 expression in WT, Rescue #22, and KO #98 ESCs (upper panel). Signal intensity of each DPPA3 band was measured relative to that of the Lamin B1 band. The lower panel shows the DPPA3 levels expressed relative to those in WT ESCs and as means ± SD (n = 3). ^*P < 0.05 by Tukey’s test. c Analysis of the DNA methylation levels of the Dppa3 promoter. Genomic DNA was isolated from WT, KO #98 and #156, and Rescue #22 and #10 ESCs. The CpG islands in the upstream region of the Dppa3 gene were analyzed for cytosine methylation. Filled and open circles represent methylated and unmethylated CpGs, respectively. The percentage of methylated CpG sites is shown under each panel. d Analysis of 5mC and 5hmC levels by dot blot assay. Control 5mC and 5hmC DNA and genomic DNA from WT, KO #98 and #156, and Rescue #22 and #10 ESCs were denatured. Two-fold serially diluted DNAs were spotted onto membranes. Antibody against 5mC (left panel) or 5hmC (right panel) was utilized for detection.