Methods in cell biology最新文献_第2页

Assessment of DNA fibers to track replication dynamics. 评估 DNA 纤维以跟踪复制动态。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-03-30 DOI: 10.1016/bs.mcb.2023.02.007

L G Bennett, C J Staples

{"title":"Assessment of DNA fibers to track replication dynamics.","authors":"L G Bennett, C J Staples","doi":"10.1016/bs.mcb.2023.02.007","DOIUrl":"10.1016/bs.mcb.2023.02.007","url":null,"abstract":"DNA replication is a complex and tightly regulated process that must proceed accurately and completely if the cell is to faithfully transmit genetic material to its progeny. Organisms have thus evolved complex mechanisms to deal with the myriad exogenous and endogenous sources of replication impediments to which the cell is subject. These mechanisms are of particular relevance to cancer biology, given that such \"replication stress\" frequently foreshadows genome instability during cancer pathogenesis, and that many traditional chemotherapies and a number of precision medicines function by interfering with the progress of DNA replication. Visualization of the progress and dynamics of DNA replication in living cells was historically a major challenge, neatly surmounted by the development of DNA fiber assays that utilize the fluorescent detection of halogenated nucleotides to track replication forks at single-molecule resolution. This methodology has been widely applied to study the dynamics of unperturbed DNA replication, as well as the cellular responses to various replication stress scenarios. In recent years, subtle modifications to DNA fiber assays have facilitated assessment of the stability of nascent DNA at stalled replication forks, as well as the detection of single-stranded DNA gaps and their subsequent filling by error-prone polymerases. Here, we present and discuss several iterations of the fiber assay and suggest methodologies for the analysis of the data obtained.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assessment of cell cycle progression and mitotic slippage by videomicroscopy. 通过视频显微镜评估细胞周期进展和有丝分裂滑动。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-08-21 DOI: 10.1016/bs.mcb.2023.03.004

Luca Mattiello, Sara Soliman Abdel Rehim, Gwenola Manic, Ilio Vitale

引用次数: 0

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors. 流式细胞仪辅助量化 CDK4/6 抑制剂治疗的癌细胞的细胞周期停滞。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-08-22 DOI: 10.1016/bs.mcb.2023.02.018

Vanessa Klapp, Norma Bloy, Carlos Jiménez-Cortegana, Aitziber Buqué, Giulia Petroni

{"title":"Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.","authors":"Vanessa Klapp, Norma Bloy, Carlos Jiménez-Cortegana, Aitziber Buqué, Giulia Petroni","doi":"10.1016/bs.mcb.2023.02.018","DOIUrl":"10.1016/bs.mcb.2023.02.018","url":null,"abstract":"Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

DNA damage and chromosomal instability. DNA 损伤和染色体不稳定性

4区生物学

Methods in cell biology Pub Date : 2024-01-01 DOI: 10.1016/S0091-679X(24)00029-3

Christian Zierhut

引用次数: 0

A new procedure to induce aortic aneurysms in mice. 诱发小鼠主动脉瘤的新程序。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-05-31 DOI: 10.1016/bs.mcb.2024.03.005

Raquel Rodrigues-Díez, Antonio Tejera-Muñoz, Raúl R Rodrigues-Diez

{"title":"A new procedure to induce aortic aneurysms in mice.","authors":"Raquel Rodrigues-Díez, Antonio Tejera-Muñoz, Raúl R Rodrigues-Diez","doi":"10.1016/bs.mcb.2024.03.005","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.03.005","url":null,"abstract":"Aortic aneurysms (AAs) are a major public health challenge, featured by a progressive impairs in aortic wall integrity that drives to aortic dilation and, in end stage, to its rupture. Despite important advances in the surgical treatment of aortic aneurysms, there is currently no pharmacological intervention that prevents their development, reduces their expansion, or avoids their rupture. In addition to classic risk factors such age or gender, several heritable connective tissue disorders have been associated with AA developing, highlighting the role of extracellular matrix (ECM) genes alterations in the developing of AA. In this sense, we have recently demonstrated that global deletion of the cellular communicating network factor 2 (CCN2), previously known as connective tissue growth factor (CTGF) due to its role in the extracellular matrix formation, predisposes to early and lethal AAs development after Angiotensin II (Ang II) infusion in mice. Here, we detail the protocol to induce and detect AAs generation in inducible global CCN2 knockout mice after Ang II infusion which allow the characterization of CCN role in AA development and may help to the development of pharmacological target for AA treatment.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Purification and analysis of kidney-infiltrating leukocytes in a mouse model of lupus nephritis. 狼疮肾炎小鼠模型中肾脏浸润白细胞的纯化与分析

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-05-01 DOI: 10.1016/bs.mcb.2024.03.007

Laura Amo, Hemanta K Kole, Bethany Scott, Francisco Borrego, Chen-Feng Qi, Hongsheng Wang, Silvia Bolland

{"title":"Purification and analysis of kidney-infiltrating leukocytes in a mouse model of lupus nephritis.","authors":"Laura Amo, Hemanta K Kole, Bethany Scott, Francisco Borrego, Chen-Feng Qi, Hongsheng Wang, Silvia Bolland","doi":"10.1016/bs.mcb.2024.03.007","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.03.007","url":null,"abstract":"Renal injury often occurs as a complication in autoimmune diseases such as systemic lupus erythematosus (SLE). It is estimated that a minimum of 20% SLE patients develop lupus nephritis, a condition that can be fatal when the pathology progresses to end-stage renal disease. Studies in animal models showed that incidence of immune cell infiltrates in the kidney was linked to pathological injury and correlated with severe lupus nephritis. Thus, preventing immune cell infiltration into the kidney is a potential approach to impede the progression to an end-stage disease. A requirement to investigate the role of kidney-infiltrating leukocytes is the development of reproducible and efficient protocols for purification and characterization of immune cells in kidney samples. This chapter describes a detailed methodology that discriminates tissue-resident leukocytes from blood-circulating cells that are found in kidney. Our protocol was designed to maximize cell viability and to reduce variability among samples, with a combination of intravascular staining and magnetic bead separation for leukocyte enrichment. Experiments included as example were performed with FcγRIIb[KO] mice, a well-characterized murine model of SLE. We identified T cells and macrophages as the primary leukocyte subsets infiltrating into the kidney during severe nephritis, and we extensively characterized them phenotypically by flow cytometry.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Animal models of disease: Achievements and challenges. 疾病动物模型：成就与挑战。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 DOI: 10.1016/S0091-679X(24)00164-X

José Manuel Bravo-San Pedro, Fernando Aranda, Aitziber Buqué, Lorenzo Galluzzi

引用次数: 0

A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine. 用于监测低甲基化胞嘧啶类似物地西他滨和阿扎胞苷治疗的药效学试验。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-21 DOI: 10.1016/bs.mcb.2024.02.016

James W Jacobberger, Philip G Woost

引用次数: 0

Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation. 通过中心粒失活诱导染色体特异性微核和染色体三分裂。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-11-28 DOI: 10.1016/bs.mcb.2022.10.009

Yu-Fen Lin, Qing Hu, Alison Guyer, Daniele Fachinetti, Peter Ly

{"title":"Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation.","authors":"Yu-Fen Lin, Qing Hu, Alison Guyer, Daniele Fachinetti, Peter Ly","doi":"10.1016/bs.mcb.2022.10.009","DOIUrl":"10.1016/bs.mcb.2022.10.009","url":null,"abstract":"Chromothripsis describes the catastrophic fragmentation of individual chromosomes followed by its haphazard reassembly into a derivative chromosome harboring complex rearrangements. This process can be initiated by mitotic cell division errors when one or more chromosomes aberrantly mis-segregate into micronuclei and acquire extensive DNA damage. Approaches to induce the formation of micronuclei encapsulating random chromosomes have been used; however, the eventual reincorporation of the micronucleated chromosome into daughter cell nuclei poses a challenge in tracking the chromosome for multiple cell cycles. Here we outline an approach to genetically engineer stable human cell lines capable of efficient chromosome-specific micronuclei induction. This strategy, which targets the CENP-B-deficient Y chromosome centromere for inactivation, allows the stepwise process of chromothripsis to be experimentally recapitulated, including the mechanisms and timing of chromosome fragmentation. Lastly, we describe the integration of a selection marker onto the micronucleated Y chromosome that enables the diverse genomic rearrangement landscape arising from micronuclei formation to be interrogated.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11008423/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Spectral analysis and sorting of microbial organisms using a spectral sorter. 使用光谱分拣机对微生物进行光谱分析和分拣。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-12 DOI: 10.1016/bs.mcb.2024.02.017

Sharath Narayana Iyengar, J Paul Robinson

引用次数: 0