The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts

Çelik-Uzuner, Selcen; Li, Yan; O’Neill, Chris

doi:10.1016/j.yexcr.2015.08.017

Cited by 7 publications

(1 citation statement)

References 61 publications

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“…To the best of knowledge, there is no study including treatment(s) with proteolytic enzymes to discard proteins from methylated (modified) DNA. In a previous study in mouse embryonic fibroblasts, trypsin was added after hydrochloric acid, which optimized conditions to reveal more methylation than compared to acid alone (Celik, Li, & O'Neill, , ). However, trypsin was not as effective in methylation staining for the detection of hydroxymethylation in mouse zygotes (Li & O'Neill, ).…”

Section: Introductionmentioning

confidence: 99%

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Çelik-Uzuner

2019

Microsc Res Tech

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Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF‐1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.

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Section: Introductionmentioning

confidence: 99%

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Çelik-Uzuner

2019

Microsc Res Tech

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Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta‐Actin in Liver and Breast Cancer Cells

Tetikoğlu

Çelik-Uzuner

2023

Chemistry & Biodiversity

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Bee venom is a natural mixture and candidate anti‐cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β‐actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β‐actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA‐MB‐231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and β‐actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of β‐actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of β‐actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β‐actin.

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Immunological Staining of Global Changes in DNA Methylation in the Early Mammalian Embryo

O’Neill

2017

Methods in Molecular Biology

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The structural complexity of chromatin can make antibody access to some nuclear antigens energetically unfavorable. This can limit the usefulness and reliability of immunostaining unless validated methods of epitope retrieval are applied. We found that denaturation of chromatin by sequential use of acidification and tryptic digestion of fixed cells is required to reliably detect DNA methylation in the embryo. Using this method to unmask the epitope revealed an unexpected pattern of reprogramming of global patterns of DNA methylation in the preimplantation embryo. This paper provides a detailed description of the procedures required for immunological detection of 5-methylcytosine in the early embryo.

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The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts

Cited by 7 publications

References 61 publications

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta‐Actin in Liver and Breast Cancer Cells

Immunological Staining of Global Changes in DNA Methylation in the Early Mammalian Embryo

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