Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells

Kilgas, Susan; Kiltie, Anne E.; Ramadan, Kristijan

doi:10.1016/j.xpro.2021.100978

Cited by 7 publications

(2 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were seeded on coverslips and treated as described in the immunofluorescence section above. The quantification of foci/cell was executed using the protocol used by Kilgas et al, 2021 . For the experiment in Figure 3—figure supplement 2 , in which cells were treated for 6 days, because of the presence of bi and multinucleation, the number of 53BP1 foci per cell was normalised according to their number of nuclei, resulting in the number of 53BP1 foci/nuclei informed.…”

Section: Methodsmentioning

confidence: 99%

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

Martino

Siri

Calzetta

et al. 2023

eLife

View full text Add to dashboard Cite

The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M-phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M-phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.

show abstract

Section: Methodsmentioning

confidence: 99%

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

Martino

Siri

Calzetta

et al. 2023

eLife

View full text Add to dashboard Cite

show abstract

“…Notably, this is the first time such a protocol has been described for RNAPII by flow cytometry. Triton X-100 treatment is performed in a sucrose-containing buffer, similar to pre-extraction/cytoskeletal buffers commonly used in immunofluorescence microscopy ( Britton et al, 2013 ; Kilgas et al, 2021 ). However, as RNAPII is known to be released from chromatin in mitosis (Parsons and Spencer, 1997), NaCl concentrations were optimized based on the expected low chromatin binding of RNAPII in mitotic cells ( Bay et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry

Bay¹,

Stokke²,

Syljuåsen³

et al. 2023

BIO-PROTOCOL

View full text Add to dashboard Cite

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5– and serine 2–phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription. Graphical overview

show abstract

The one‐carbon metabolic enzyme MTHFD2 promotes resection and homologous recombination after ionizing radiation

Marttila,

Bonagas,

Chalkiadaki

et al. 2024

Molecular Oncology

View full text Add to dashboard Cite

The one‐carbon metabolism enzyme bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is among the most overexpressed proteins across tumors and is widely recognized as a promising anticancer target. While MTHFD2 is mainly described as a mitochondrial protein, a new nuclear function is emerging. Here, we observe that nuclear MTHFD2 protein levels and association with chromatin increase following ionizing radiation (IR) in an ataxia telangiectasia mutated (ATM)‐ and DNA‐dependent protein kinase (DNA‐PK)‐dependent manner. Furthermore, repair of IR‐induced DNA double‐strand breaks (DSBs) is delayed upon MTHFD2 knockdown, suggesting a role for MTHFD2 in DSB repair. In support of this, we observe impaired recruitment of replication protein A (RPA), reduced resection, decreased IR‐induced DNA repair protein RAD51 homolog 1 (RAD51) levels and impaired homologous recombination (HR) activity in MTHFD2‐depleted cells following IR. In conclusion, we identify a key role for MTHFD2 in HR repair and describe an interdependency between MTHFD2 and HR proficiency that could potentially be exploited for cancer therapy.

show abstract

Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells

Cited by 7 publications

References 20 publications

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry

The one‐carbon metabolic enzyme MTHFD2 promotes resection and homologous recombination after ionizing radiation

Contact Info

Product

Resources

About