Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: A novel method suitable for examining dynamic changes in proteins during mitotic progression

Matsui, Yoshihiko; Nakayama, Yuji; Okamoto, Mai; Fukumoto, Yasunori; Yamaguchi, Naoto

doi:10.1016/j.ejcb.2011.12.008

Cited by 52 publications

(36 citation statements)

References 36 publications

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“…To exclude the possibility that SU6656 and PP2 may nonspecifically inhibit other kinases to delay checkpoint recovery, the involvement of SFKs was addressed by RNAi-mediated gene knockdown. In HeLa S3 cells, at least three family members, Lyn, c-Yes, and Fyn, are abundantly expressed and might have considerable functional redundancy (64). Because previous studies reported the involvement of Lyn in DNA damage responses (44,45), we examined the role of Lyn.…”

Section: Sfk Activity Is Required For G 2 Dna Damage Checkpointmentioning

confidence: 99%

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

Fukumoto

Morii

Miura

et al. 2014

Journal of Biological Chemistry

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Background: Once DNA repair is completed, the DNA damage checkpoint is terminated, and the cell cycle is resumed. Results: Src inhibition induced a delay in G 2 checkpoint recovery and persistent ATR-Chk1 activation. Conclusion: Src inhibits ATR signaling to promote recovery from G 2 checkpoint arrest. Significance: Src sends a termination signal between the completion of DNA repair and the initiation of checkpoint termination.

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Section: Sfk Activity Is Required For G 2 Dna Damage Checkpointmentioning

confidence: 99%

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

Fukumoto

Morii

Miura

et al. 2014

Journal of Biological Chemistry

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“…We used HeLa S3, a HeLa variant cell line that can be highly synchronized in various stages of the cell cycle (26,(31)(32)(33). Treatment with mimosine at a concentration of 0.5 mM for 24 h synchronized only 55% of the cells in G 1 phase and the rest in S phase (33%) and G 2 /M phase (13%) (Fig.…”

Section: Enrichment Of Cells Having G 1 Phase Dna Content Uponmentioning

confidence: 99%

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Kubota

Fukumoto

Ishibashi

et al. 2014

Journal of Biological Chemistry

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Background: Mimosine is a cell synchronization reagent used for arresting cells in late G 1 and S phases. Results: Replication fork assembly is reversibly blocked by ATM activation through mimosine-generated reactive oxygen species. Conclusion: Mimosine induces cell cycle arrest strictly at the G 1 -S phase boundary, which prevents replication fork stallinginduced DNA damage. Significance: These findings provide a novel mechanism of the mimosine-induced G 1 checkpoint.

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“…Blebbistatin or BDM treatment extends the culture lifetime of mouse cardiac myocytes, a cell line that is notoriously difficult to maintain in primary culture and very valuable for the characterization of heart disorders [70,71]. Furthermore, selective treatment with blebbistatin has been demonstrated as a tool to synchronize mammalian culture cells during different stages of mitotic division (metaphase, anaphase, telophase), a task that proved difficult in the past due to the short duration of anaphase [72]. In addition, both BDM and blebbistatin have been used to eliminate the 'motion artifacts' caused by muscle contractions during live optical imaging of cardiac electrical activity [73,74].…”

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confidence: 99%

Small-Molecule Inhibitors of Myosin Proteins

et al. 2012

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Advances in screening and computational methods have enhanced recent efforts to discover/ design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design.The identification and characterization of pharmacological compounds that inhibit the functional activity of one or more specific proteins or processes has been the subject of much scientific investigation. On a basic science level, these membrane-permeable compounds provide the scientific community with a tool for the targeted and functional inhibition of a given protein in the cell; a potent means of evaluating the intracellular functions of that protein [1,2]. From a biomedical standpoint, the characterization of these small-molecule inhibitors affords an opportunity for the development of novel disease treatments centering on the repression of an offensive molecule or the reversal of its downstream effects [3][4][5].At present, several complementary methods for obtaining suitable small-molecule inhibitors of specific proteins exist. Traditional methods in inhibitor discovery involve the systematic testing of a series of chemically synthesized or naturally occurring compounds. Advances in robotics and data processing have made it possible to use high-throughput screens to test libraries of thousands or even millions of potential drugs for their ability to inhibit the function of a specific protein in a targeted biochemical or cellular assay [6][7][8]. These inhibitor discovery processes are complemented by more precise methods in small-molecule inhibitor design. Structure-based methods rely on the use of x-ray crystallographic or NMRbased structures of a protein of interest to design small molecules likely to bind and inhibit protein function [9,10]. Computer-aided inhibitor design uses computational methods to optimize potential inhibitors identified by screening or structure-based methods, to virtually © 2013 Folma Buss * Author for correspondence: Tel.: +44 1223 763348, Fax: +44 1223 762640, fb207@cam.ac.uk. Financial & competing interests disclosureThe authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance w...

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Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: A novel method suitable for examining dynamic changes in proteins during mitotic progression

Cited by 52 publications

References 36 publications

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Small-Molecule Inhibitors of Myosin Proteins

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