-
PloS One Mar 2011It was recently shown that cellular turnover occurs within the human adipocyte population. Through three independent experimental approaches--dilution of an inducible...
It was recently shown that cellular turnover occurs within the human adipocyte population. Through three independent experimental approaches--dilution of an inducible histone 2B-green fluorescent protein (H2BGFP), labeling with the cell cycle marker Ki67 and incorporation of BrdU--we characterized the degree of cellular turnover in murine adipose tissue. We observed rapid turnover of the adipocyte population, finding that 4.8% of preadipocytes are replicating at any time and that between 1-5% of adipocytes are replaced each day. In light of these findings, we suggest that adipose tissue turnover represents a possible new avenue of therapeutic intervention against obesity.
Topics: Adipocytes; Adipose Tissue; Animals; Bromodeoxyuridine; Cell Cycle; Cell Proliferation; Humans; Ki-67 Antigen; Mice; Obesity
PubMed: 21407813
DOI: 10.1371/journal.pone.0017637 -
STAR Protocols Dec 2021DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3'-single-stranded DNA (3'-ssDNA). The extent of resection regulates DNA double-strand break...
DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3'-single-stranded DNA (3'-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2'-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).
Topics: Bromodeoxyuridine; Cell Line, Tumor; DNA, Single-Stranded; Genomic Instability; Humans; Microscopy, Fluorescence
PubMed: 34888531
DOI: 10.1016/j.xpro.2021.100978 -
PloS One 2014Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to...
Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2'-deoxyuridine (EdU) and 5-Chloro-2'-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2'-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry.
Topics: Bromodeoxyuridine; Cell Cycle; Cell Proliferation; DNA Replication; DNA, Fungal; Deoxyuridine; Schizosaccharomyces; Staining and Labeling; Thymidine
PubMed: 24551125
DOI: 10.1371/journal.pone.0088629 -
Redox Report : Communications in Free... Dec 2022Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus. Oxidative stress resulting from high glucose promotes accumulation...
OBJECTIVES
Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus. Oxidative stress resulting from high glucose promotes accumulation of ECM and development of DN. Activation of Nrf2 could attenuate oxidative stress and following accumulation of ECM. To find novel therapy for DN, we explored the effects of swinhoeic acid from on mesangial cells under high glucose and underlying mechanisms.
METHODS
CCK-8 and BrdU incorporation assays for survival of mesangial cells gave the concentration of swinhoeic acid in following investigations. ROS, MDA, SOD and CAT were determined. And ECM proteins and their upstream regulators TGF-β and CTGF were detected using ELISA assays. Activation of Nrf2 was explored by immunofluorescence staining together with luciferase reporter assay. To demonstrate the role of Nrf2 activation, siRNA interference was performed. And co-immunoprecipitation assay was used to elucidate swinhoeic acid affects the interaction between Keap1 and Nrf2.
RESULTS
Swinhoeic acid at 10 and 20 μM attenuated oxidative stress and accumulation of ECM in mesangial cells under high glucose. Itactivated Nrf2 in a Keap1-dependent manner, which was involved in its effects.
CONCLUSION
Swinhoeic acid ameliorates oxidative stress and accumulation of ECM resulting from high glucose in mesangial cells via activating Nrf2 in Keap1-dependent manner.
Topics: Kelch-Like ECH-Associated Protein 1; Mesangial Cells; NF-E2-Related Factor 2; Potentilla; Reactive Oxygen Species; RNA, Small Interfering; Sincalide; Bromodeoxyuridine; Signal Transduction; Oxidative Stress; Diabetic Nephropathies; Glucose; Superoxide Dismutase; Extracellular Matrix
PubMed: 36259553
DOI: 10.1080/13510002.2022.2134755 -
Methods in Molecular Biology (Clifton,... 2019We recently developed a method for assessing RNA-DNA interactions using proximity ligation assays (PLA). This technique, termed the "RNA-DNA interaction assay" (RDIA),...
We recently developed a method for assessing RNA-DNA interactions using proximity ligation assays (PLA). This technique, termed the "RNA-DNA interaction assay" (RDIA), involves differentially labeling DNA and RNA with EdU and BrU, respectively. Once labeled, PLA is performed to assess if the labeled molecules are in close proximity. Here we provide a detailed description of the modified RDIA protocol utilizing currently commercially available BrdU antibodies. As an example, we show its ability to detect nascent transcripts on recently synthesized DNA in both cultured H1299 cells and mouse embryonic stem cells.
Topics: Animals; Antibodies; Bromodeoxyuridine; Cell Line; DNA; Humans; Mice; Mouse Embryonic Stem Cells; RNA
PubMed: 31124093
DOI: 10.1007/978-1-4939-9537-0_10 -
Neoplasia (New York, N.Y.) Aug 2008The thymidine analog bromodeoxyuridine (BrdU) is incorporated into newly synthesized DNA and has been shown to increase the susceptibility of incorporating cells to...
The thymidine analog bromodeoxyuridine (BrdU) is incorporated into newly synthesized DNA and has been shown to increase the susceptibility of incorporating cells to ionizing radiation. However, in the absence of secondary stressors, BrdU is thought to substitute relatively benignly for thymidine and is commonly used to "birth-date" proliferative cells. We report a novel antiproliferative effect of BrdU on cancer cells, which is independent of its role in radiosensitization. A single, brief in vitro exposure to BrdU induces a profound and sustained reduction in the proliferation rate of all cancer cells examined. Cells do not die but variably up-regulate some senescence-associated proteins as they accumulate in the G1 phase of the cell cycle. Bromodeoxyuridine also impairs the proliferative capacity of primary tumor-initiating human glioma cells and may therefore represent a means of targeting cancer stem cells. Finally, conservative in vivo BrdU regimens--in the absence of any other treatment--significantly suppress the progression of gliomas in the highly aggressive, syngeneic RG2 model. These results suggest that BrdU may have an important role as an adjunctive therapeutic for a wide variety of cancers based on new insights into its effect as a negative regulator of cell cycle progression.
Topics: Administration, Oral; Animals; Bromodeoxyuridine; Cell Cycle; Cell Proliferation; Disease Progression; Glioma; Humans; Injections, Intraperitoneal; Injections, Subcutaneous; Male; Neoplasms, Experimental; Nucleosides; Rats; Rats, Inbred F344; Time Factors; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 18680882
DOI: 10.1593/neo.08382 -
Nature Protocols Jun 2011Replication timing profiles are cell type-specific and reflect genome organization changes during differentiation. In this protocol, we describe how to analyze...
Replication timing profiles are cell type-specific and reflect genome organization changes during differentiation. In this protocol, we describe how to analyze genome-wide replication timing (RT) in mammalian cells. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) and sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated, amplified, differentially labeled and co-hybridized to a whole-genome comparative genomic hybridization microarray, which is currently more cost effective than high-throughput sequencing and equally capable of resolving features at the biologically relevant level of tens to hundreds of kilobases. We also present a guide to analyzing the resulting data sets based on methods we use routinely. Subjects include normalization, scaling and data quality measures, LOESS (local polynomial) smoothing of RT values, segmentation of data into domains and assignment of timing values to gene promoters. Finally, we cover clustering methods and means to relate changes in the replication program to gene expression and other genetic and epigenetic data sets. Some experience with R or similar programming languages is assumed. All together, the protocol takes ∼3 weeks per batch of samples.
Topics: Bromodeoxyuridine; Comparative Genomic Hybridization; Computational Biology; DNA Replication; Flow Cytometry; Genomics; Immunoprecipitation; Nucleic Acid Amplification Techniques; S Phase; Software
PubMed: 21637205
DOI: 10.1038/nprot.2011.328 -
Cytometry Nov 1985The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our...
The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Bromodeoxyuridine; Cell Line; Cricetinae; Cricetulus; Enzyme-Linked Immunosorbent Assay; Female; Ovary
PubMed: 4064836
DOI: 10.1002/cyto.990060603 -
The Journal of Neuroscience : the... Mar 1999The mitotic marker 5-bromodeoxyuridine (BrdU) was injected twice daily (60 mg/kg) into pregnant hooded rats on one of embryonic days (E) 11, 12, 13, 15, 17, or 21, or...
The mitotic marker 5-bromodeoxyuridine (BrdU) was injected twice daily (60 mg/kg) into pregnant hooded rats on one of embryonic days (E) 11, 12, 13, 15, 17, or 21, or into rat pups on postnatal day (P) 10. The principal findings were the following: (1) BrdU exposure on E11 produces profound effects on body morphology, and animals must be fed a special diet because of chronic tooth abnormalities; (2) BrdU exposure at E17 or earlier produces a change in coat spotting pattern, the precise pattern varying with age; (3) BrdU exposure on E15 or earlier produces a reduction in both brain and body weight; (4) BrdU exposure on E17 or earlier reduces cortical thickness; (5) BrdU exposure on E11-E13 and at P10 reduces cerebellar size relative to cerebral size; (6) spatial learning is significantly affected after injections of BrdU at E11-E17, but the largest effect is on E17; (7) the deficit in spatial learning may be related in part to a reduction in visual acuity; and (8) skilled forelimb ability is most disrupted after BrdU exposure at E15 but is also impaired after injections on E13 or earlier. BrdU thus has teratological effects on body, brain, and behavior that vary with the developmental age of the fetus or infant.
Topics: Abnormalities, Drug-Induced; Aging; Animals; Animals, Newborn; Behavior, Animal; Behavioral Symptoms; Brain; Bromodeoxyuridine; Drug Administration Schedule; Embryo, Mammalian; Injections; Rats; Rats, Long-Evans
PubMed: 10066283
DOI: 10.1523/JNEUROSCI.19-06-02337.1999 -
The International Journal of... 2022Even before the first synapses appear, neurotransmitters and their receptors are present in the developing brain, regulating the cell fate of neuronal progenitors in...
Even before the first synapses appear, neurotransmitters and their receptors are present in the developing brain, regulating the cell fate of neuronal progenitors in neurogenic niches, such as the lateral ventricle. In particular, dopamine appears to play a pivotal role in the neurogenesis of the subventricular zone by controlling the proliferation and differentiation of progenitors through activation of different receptors. Although dopamine receptor 5 (D5R) is expressed prenatally, there is little information regarding its role in either pre- or postnatal forebrain development. To examine the role of D5Rs in neurogenesis in the rat lateral ventricle subventricular zone (V-SVZ), we immunohistochemically defined D5R expression, as well as BrdU incorporation in progenitor cells of various post-weaning stages (Post-natal day (P) 20 until P80). We found that the level of proliferating cells is stable from postnatal day 20 until 50, and then declines sharply on P80. Concomitantly, D5R is expressed in all ages examined, but we detected a progressive decrease in the density of D5R+ cells from P40 until P80. Moreover, double immunostaining for BrdU and D5R revealed that proliferating cells in V-SVZ also express D5R. Collectively, our data suggest that D5R is expressed in the post-weaning V-SVZ of rat at least until P80, and its expression pattern coincides with that of proliferating cells in the V-SVZ, hinting at a possible role of D5Rs in the regulation of neuronal progenitor division/differentiation.
Topics: Animals; Bromodeoxyuridine; Cell Differentiation; Cell Proliferation; Dopamine; Lateral Ventricles; Neurogenesis; Rats; Receptors, Dopamine D5; Weaning
PubMed: 34881789
DOI: 10.1387/ijdb.210163as