The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage

Oeck, Sebastian; Malewicz, Nathalie M.; Hurst, Sebastian; Rudner, Justine; Jendrossek, Verena

doi:10.1186/s13014-015-0453-1

Cited by 51 publications

(49 citation statements)

References 41 publications

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“…Images were acquired with a Nikon fluorescence microscope, and data were collected and analyzed with NIS software (Nikon). For 53BP1 foci, their number per nucleus was determined using the Focinator tool (Oeck, Malewicz, Hurst, Rudner, & Jendrossek, 2015). More than 200 nuclei were analyzed for each condition.…”

Section: Methodsmentioning

confidence: 99%

The nuclear receptor RXRA controls cellular senescence by regulating calcium signaling

Warnier

Raynard

et al. 2018

Aging Cell

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Calcium signaling is emerging as a key pathway controlling cellular senescence, a stable cell proliferation arrest playing a fundamental role in pathophysiological conditions, such as embryonic development, wound healing, cancer, and aging. However, how calcium signaling is regulated is still only partially understood. The inositol 1, 4, 5‐trisphosphate receptor type 2 (ITPR2), an endoplasmic reticulum calcium release channel, was recently shown to critically contribute to the implementation of senescence, but how ITPR2 expression is controlled is unclear. To gain insights into the regulation of ITPR2 expression, we performed an siRNA screen targeting 160 transcription factors and epigenetic regulators. Interestingly, we discovered that the retinoid X receptor alpha (RXRA), which belongs to the nuclear receptor family, represses ITPR2 expression and regulates calcium signaling though ITPR2 and the mitochondrial calcium uniporter (MCU). Knockdown of RXRA induces the production of reactive oxygen species (ROS) and DNA damage via the ITPR2‐MCU calcium signaling axis and consequently triggers cellular senescence by activating p53, whereas RXRA overexpression decreases DNA damage accumulation and then delays replicative senescence. Altogether, our work sheds light on a novel mechanism controlling calcium signaling and cellular senescence and provides new insights into the role of nuclear receptors.

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Section: Methodsmentioning

confidence: 99%

The nuclear receptor RXRA controls cellular senescence by regulating calcium signaling

Warnier

Raynard

et al. 2018

Aging Cell

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“…A minimum of 120 cells were imaged per sample. Images were analyzed by counting the number of γ-H2AX foci using Focinator software (21), and noise tolerance level was set to 80. Cells with >5 foci in the nucleus were considered to be γ-H2AX foci-positive.…”

Section: Methodsmentioning

confidence: 99%

Apoptosis is augmented in high-grade serous ovarian cancer by the combined inhibition of Bcl-2/Bcl-xL and PARP

Yokoyama

Kohn

Brill

et al. 2017

International Journal of Oncology

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The aim of our study was to evaluate possible synergistic cytotoxic effects of the combination treatment with the BH3-mimetic ABT-263 and the PARP inhibitor BMN 673 in high-grade serous ovarian cancer (HGSOC) cells using clinically achievable concentrations of each drug. In vitro cytotoxic effects of ABT-263 and BMN 673 were assessed by XTT assay in three HGSOC cell lines: OVCAR3, OVCAR8, and OV90 cells. Combination index values and synergy/antagonism volumes were used to determine synergy. The drug effects on DNA damage accumulation, cell cycle progression, apoptosis induction, and expression levels of Bcl-2 family proteins were examined to dissect molecular mechanisms. The combination treatment synergistically decreased cell viability in a concentration- and time-dependent manner in all cell lines; combination index values were <0.9 and synergy/antagonism volumes were >100 after 72 h of treatment. Clinically achievable concentrations of ABT-263 2 µM and BMN 673 25 nM were used to investigate mechanisms. No increase in γ-H2AX foci formation was observed with addition of ABT-263 to BMN 673 treatment. The combination treatment increased the sub-G1 and Annexin V-positive cell populations after 48 h compared with the control and each monotherapy. It also induced greater caspase-3/7 activity and PARP cleavage. ABT-263 alone and in combination with BMN 673 induced expression levels of Bim, a pro-apoptotic protein. In conclusion, the ABT-263 and BMN 673 combination resulted in synergistic cytotoxic effects against HGSOC cells through greater induction of apoptosis. This may be a novel therapeutic strategy for HGSOC.

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“…All wells were imaged using a high throughput program in the CV1000 Acquisition software, allowing for 4 images per well to be taken with multiple confocal planes and computational autofocus. MIP files were then utilized for analysis by R-package Focinator v2 77 . In brief, the number of foci were counted within each nucleus, excluding nuclei on edge of each frame.…”

Section: Discussionmentioning

confidence: 99%

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

Guard

Poss

Ebmeier

et al. 2018

Preprint

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Loss of function mutations in the protein kinase DYRK1A lead to a syndromic form of autism spectrum disorder and intellectual disability. Conversely, increased DYRK1A dosage is implicated in atypical brain development and neurocognitive deficits in trisomy 21. DYRK1A regulates a diverse array of cellular processes through kinase dependent and independent interactions with substrates and binding partners. Recent evidence implicates DYRK1A in direct regulation of the transcriptional machinery, but many of the molecular details are not yet known.Furthermore, the landscape of DYRK1A interactions in the nucleus is incomplete, impeding progress toward understanding its function in transcription. Here, we used immunoaffinity purification and mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. These were enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage. We further show that knockout of DYRK1A or treatment with DYRK1A inhibitors in HeLa cells impaired efficient recruitment of 53BP1 to DNA double strand breaks induced by ionizing radiation. This nuclear interactome thus reveals a new role for DYRK1A in DNA damage repair and provides a resource for exploring new functions of DYRK1A in the nucleus. MAIN BODY

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The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage

Cited by 51 publications

References 41 publications

The nuclear receptor RXRA controls cellular senescence by regulating calcium signaling

The nuclear receptor RXRA controls cellular senescence by regulating calcium signaling

Apoptosis is augmented in high-grade serous ovarian cancer by the combined inhibition of Bcl-2/Bcl-xL and PARP

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

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