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. 2023 Feb 27;15:29. doi: 10.1186/s13148-023-01446-4

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© The Author(s) 2023

Open AccessThis 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 licence, and indicate if changes were made. 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/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — DNA methylation differs in cis-regulatory regions in c-Jun gene targets. A Heatmap of hierarchical clustering analysis showing median DNA methylation of promoters of c-Jun target genes (2 kb upstream and 500 bp downstream relative to TSS) in glioma samples. The following labels were used: IDHmut (4 GII/GIII tumors), GII/GIII (4 only GII/GIII-IDHwt tumors) and GIV (10 IDHwt tumors). DNA methylation levels showed as beta values, with 0.0–0.2 representing hypomethylated cytosines and 0.6–1.0 representing hypermethylated cytosines. B Differences in beta values distribution in gene promoters with the predicted c-Jun TFBS in GII/GIII-IDHwt versus GIV glioma samples that are statistically non-significant (upper panel) and statistically significant (bottom panel). C Distance of c-Jun motif to the beginning of differentially methylated C-rich regions between high- and low-grade gliomas. Green boxes represent a c-Jun predicted binding site, while brown boxes show each C-rich region that was found significantly differently methylated between low- and high-grade glioma samples (Chi-squared test at significance level adjusted p < 0.05). D Competitive electrophoretic mobility assay (EMSA) was used for measuring binding affinity of nuclear extracts from tumor derived cell lines and NHA, to the methylated and unmethylated double-stranded DNA from the UPP1 promoter site. Binding of proteins from NHA, WG12, LN18, and LN229 to the methylated and unmethylated probes was evaluated. Lane 1, 6: unlabeled probes; lanes 2–5 and 7–10: protein binding. E Probe binding strength in EMSA densitometry analysis is expressed as a percentage of the overall variation across all cell lines. One-way ANOVA with Fisher's LSD post hoc tests revealed significant differences at *p < 0.05 and **p < 0.01; Hedge's effect size (g) indicates the strength of the difference of the binding between the studied cell lines (medium effects g ≈ 0.5; large effects g ≈ 0.8), n = 3 ± SD