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. 2015 Oct 16;7(3):2433–2454. doi: 10.18632/oncotarget.5693

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Copyright: © 2016 Uziel et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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A. Single stranded (SS) overhang length in BRCA1/2 carriers vs control. B. SS overhang after BRCA1 silencing in HB-2 breast epithelial cells. The length of the SS overhang was measured in both systems as described in the Method section by the luminescence obtained from the binding of a PNA probe to the DNA isolated from all samples and measured by luminometer. WT- the wild type intact HB-2 cells, NC- HB-3 cells transfected with a scrambled negative control plasmid. C. Levels of DNMT-1 in BRCA1/2 mutations carriers. The upper panel is an example of one Western blot and the lower is a quantitation of three independent experiments. Telomere structure, sub-telomeric methylation and the shelterin proteins binding to telomeres. D. Levels of DNMT-1 after BRCA1 silencing. The upper panel is an example of one Western blot and the lower is a quantitation of three independent experiments. “NC” depicts the cells which were transfected with the negative control shRNA plasmid; “4” “ is clone # 4 in which BRCA1 silencing was optimal one month post shRNA transfection. “4” is clone # 4 in which BRCA1 silencing was optimal 6 months post shRNA transfection. E. Binding of the shelterin members to telomeres. Cells were subjected to the ChIP assay analysis using specific shelterin proteins antibodies and a DIG- telomere complementary probe. The upper panel contains a representative ChIP-dot blot assay and the lower is a quantitation of three independent ChIP assays. “WT” - the wild type intact cells; “NC” - cells transfected with the negative control shRNA plasmid; “4” - clone # 4 in which BRCA1 silencing was optimal one month post shRNA transfection. “4”- clone # 4 in which BRCA1 silencing was optimal 6 months post shRNA transfection.

A. Single stranded (SS) overhang length in BRCA1/2 carriers vs control. B. SS overhang after BRCA1 silencing in HB-2 breast epithelial cells. The length of the SS overhang was measured in both systems as described in the Method section by the luminescence obtained from the binding of a PNA probe to the DNA isolated from all samples and measured by luminometer. WT- the wild type intact HB-2 cells, NC- HB-3 cells transfected with a scrambled negative control plasmid. C. Levels of DNMT-1 in BRCA1/2 mutations carriers. The upper panel is an example of one Western blot and the lower is a quantitation of three independent experiments. Telomere structure, sub-telomeric methylation and the shelterin proteins binding to telomeres. D. Levels of DNMT-1 after BRCA1 silencing. The upper panel is an example of one Western blot and the lower is a quantitation of three independent experiments. “NC” depicts the cells which were transfected with the negative control shRNA plasmid; “4” “ is clone # 4 in which BRCA1 silencing was optimal one month post shRNA transfection. “4” is clone # 4 in which BRCA1 silencing was optimal 6 months post shRNA transfection. E. Binding of the shelterin members to telomeres. Cells were subjected to the ChIP assay analysis using specific shelterin proteins antibodies and a DIG- telomere complementary probe. The upper panel contains a representative ChIP-dot blot assay and the lower is a quantitation of three independent ChIP assays. “WT” - the wild type intact cells; “NC” - cells transfected with the negative control shRNA plasmid; “4” - clone # 4 in which BRCA1 silencing was optimal one month post shRNA transfection. “4”- clone # 4 in which BRCA1 silencing was optimal 6 months post shRNA transfection.