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. 2019 Jan 24;9:594. doi: 10.1038/s41598-018-37444-3

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© The Author(s) 2019

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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Loss of heterochromatin and DDR in cortical neurons of Bmi1^+/− mice. (a) Western blot analysis of cortical extracts from old WT (n = 6) and Bmi1^+/− (n = 6) mice. (b) IHC on cortical sections from old WT (n = 3) and Bmi1^+/− (n = 3) mice with quantification of the number of H3K9^me3 foci per nucleus (6 sections/sample were counted). Scale bar in the inset: 3μm. (c) Real-time RT-PCR analysis for the expression of satellite repeats in cortices from WT (n = 3) and Bmi1^+/− (n = 3) mice. (d) Western blot analysis of cortical extracts from old WT (n = 3) and Bmi1^+/− (n = 3) mice. (e) IHC for γH2AX on cortical sections of old WT and Bmi1^+/− mice. (f) ChIP analyses were performed on frontal cortex homogenates from 15 month-old WT (n = 3) and Bmi1^+/− (n = 3) mice. Quantitative PCR was performed in triplicate for each DNA sequence. All data are presented as fold of input. Bmi1 and H2A^ub reduction (p < 0.05), and p-ATR and γH2AX accumulation (p < 0.01) in Bmi1^+/− mice are significant (Two-way ANOVA analysis). (bottom panel) Fold differences for γH2AX and p-ATR accumulation in Bmi1^+/− mice were measured relative to WT levels for each DNA sequences. (g) Cortical slices from 45-day old mice. Areas showed are located in layers 2-3 of the frontal cortex. In WT samples, large pyramidal neurons were strongly labeled by the H3K9^me3 antibody and H3K9^me3 co-localized with DAPI. In Bmi1^+/− samples, large pyramidal neurons were weakly labeled by the H3K9^me3 antibody and the H3K9me3 signal was fragmented. Bottom panels: Quantification of the images showed in (f). Values are mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001; Student’s t-test.