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. 2018 Jul 18;8:10888. doi: 10.1038/s41598-018-29239-3

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© The Author(s) 2018

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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MDC1 methylation is required for the amplification of activated ATM around the DNA double strand break sites. (a) Coimmunoprecipitation of MDC1 with ATM, NBS1 or MRE11 from U2OS cells transfected with HA-tagged MDC1 (WT or K45A). Asterisk: non-specific bands. Right panels: Quantification of immunoprecipitated ATM, NBS1 and MRE11 calculated from the signal intensities normalized against level of MDC1 immunoprecipitates. Error bars, SD from triplicate measurements. (b,c) Immunofluorescence analysis of U2OS cells transfected with indicated siRNA oligonucleotides, (b) and siRNA-resistant HA-tagged MDC1 (WT or K45A), using indicated antibodies at 2 h after exposure to NCS (50 ng/ml for 15 min). Scale bar, 10 μm. (d) Immunoblotting analysis of whole-cell extracts from U2OS cells transfected with control, EHMT1- or EHMT2-targeting siRNAs, using the indicated antibodies at 2 h after exposure to NCS (100 ng/ml for 30 min). (e) In situ PLA of RPE1 cells introduced with mock or TRF2-DN combined with or without the knockdown of EHMT1 or EHMT2, subjected to immunoreaction using TRF1 antibodies combined with MDC1 or phosphor-ser1981 ATM antibodies. Representative images are shown in the left panel. Localization of MDC1 or activated ATM at dysfunctional telomeres are quantified as the percentage of cells with more than 5 PLA signals in nuclei, each based on at least 150 cells from three independent experiments (right). Error bars represent SD. Statistical significance was calculated using two-tailed, unpaired t-test compared with control cells introduced with TRF2-DN; *P < 0.0005, **P < 0.0001. Scale bar, 10 μm.