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Current Molecular Medicine 2024Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However,...
BACKGROUND
Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However, the exact mechanism is not clear.
OBJECTIVE
This study focused on the role of CTBP1 in lung fibroblast function, elaborated its regulation mechanism, and analyzed the relationship between CTBP1 and ZEB1. Meanwhile, the antipulmonary fibrosis effect and its molecular mechanism of Toosendanin were studied.
METHODS
Human IPF fibroblast cell lines (LL-97A and LL-29) and normal fibroblast cell lines (LL-24) were cultured . The cells were stimulated with FCS, PDGF-BB, IGF-1, and TGF-β1, respectively. BrdU detected cell proliferation. The mRNA expression of CTBP1 and ZEB1 was detected by QRT-PCR. Western blotting was used to detect the expression of COL1A1, COL3A1, LN, FN, and α-SMA proteins. An animal model of pulmonary fibrosis was established to analyze the effects of CTBP1 silencing on pulmonary fibrosis and lung function in mice.
RESULTS
CTBP1 was up-regulated in IPF lung fibroblasts. Silencing CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts. Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts. Silencing CTBP1 reduced the degree of pulmonary fibrosis in mice with pulmonary fibrosis. Western blot, CO-IP, and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts. Toosendanin can inhibit the ZEB1/CTBP1protein interaction and further inhibit the progression of pulmonary fibrosis.
CONCLUSION
CTBP1 can promote the activation and proliferation of lung fibroblasts through ZEB1. CTBP1 promotes lung fibroblast activation through ZEB1, thereby increasing excessive deposition of ECM and aggravating IPF. Toosendanin may be a potential treatment for pulmonary fibrosis. The results of this study provide a new basis for clarifying the molecular mechanism of pulmonary fibrosis and developing new therapeutic targets.
Topics: Humans; Mice; Animals; Bromodeoxyuridine; Idiopathic Pulmonary Fibrosis; Lung; Fibroblasts; Transforming Growth Factor beta1; Transcription Factors; Intercellular Signaling Peptides and Proteins; Zinc Finger E-box-Binding Homeobox 1
PubMed: 37138491
DOI: 10.2174/1566524023666230501205149 -
Genes Feb 2022Eukaryotes duplicate their chromosomes during the cell cycle S phase using thousands of initiation sites, tunable fork speed and megabase-long spatio-temporal...
Eukaryotes duplicate their chromosomes during the cell cycle S phase using thousands of initiation sites, tunable fork speed and megabase-long spatio-temporal replication programs. The duration of S phase is fairly constant within a given cell type, but remarkably plastic during development, cell differentiation or various stresses. Characterizing the dynamics of S phase is important as replication defects are associated with genome instability, cancer and ageing. Methods to measure S-phase duration are so far indirect, and rely on mathematical modelling or require cell synchronization. We describe here a simple and robust method to measure S-phase duration in cell cultures using a dual EdU-BrdU pulse-labeling regimen with incremental thymidine chases, and quantification by flow cytometry of cells entering and exiting S phase. Importantly, the method requires neither cell synchronization nor genome engineering, thus avoiding possible artifacts. It measures the duration of unperturbed S phases, but also the effect of drugs or mutations on it. We show that this method can be used for both adherent and suspension cells, cell lines and primary cells of different types from human, mouse and . Interestingly, the method revealed that several commonly-used cancer cell lines have a longer S phase compared to untransformed cells.
Topics: Animals; Bromodeoxyuridine; Cell Division; Chromosomes; Flow Cytometry; Mice; S Phase
PubMed: 35327961
DOI: 10.3390/genes13030408 -
International Journal of Experimental... Jun 2022Metoclopramide (MCP) is a drug that has been widely used in recent years due to its hyperprolactinaemia effect on mothers during breastfeeding. The aim of this study was...
Metoclopramide (MCP) is a drug that has been widely used in recent years due to its hyperprolactinaemia effect on mothers during breastfeeding. The aim of this study was to investigate the proliferative changes that MCP may cause in the maternal breast tissue. In this study, 18 Wistar albino young-adult breastfeeding mothers with their offspring were divided into three groups: control group, low-dose MCP-applied group and high-dose MCP-applied group. The experiment was carried out during the lactation period and at the end of 21 days. Prolactin, BrdU and Ki-67 breast tissue distributions were evaluated by immunohistochemistry, and tissue levels were evaluated biochemically by the ELISA method. According to ELISA and immunohistochemistry results in breast tissue, there was no significant difference between Ki-67 and BrdU results in all groups. Metoclopramide did not change the expression of proliferation molecules Ki-67 and BrdU in breast tissue. These results suggested that while metoclopramide increases breast proliferation, it does not have the risk of transforming the tissue into a tumour.
Topics: Bromodeoxyuridine; Cell Proliferation; Female; Humans; Ki-67 Antigen; Lactation; Metoclopramide
PubMed: 35243705
DOI: 10.1111/iep.12433 -
Current Issues in Molecular Biology Jul 2001Measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. Protocols for DNA measurement have been developed including... (Review)
Review
Measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. Protocols for DNA measurement have been developed including Bivariate cytokeratin/DNA analysis, Bivariate BrdU/DNA analysis, and multiparameter flow cytometry measurement of cellular DNA content. This review summarises the methods for measurement of cellular DNA and analysis of the cell cycle and discusses the commercial software available for these purposes.
Topics: Antimetabolites; Bromodeoxyuridine; Cell Cycle; DNA; Flow Cytometry; Software
PubMed: 11488413
DOI: No ID Found -
Cell Research Oct 2015
Topics: Animals; Bromodeoxyuridine; Cell Line; Cellular Reprogramming; Fibroblasts; Induced Pluripotent Stem Cells; Mice
PubMed: 26251165
DOI: 10.1038/cr.2015.96 -
Methods in Molecular Biology (Clifton,... 2018Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell...
Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2'-deoxyuridine (BrdU) or tritiated thymidine ([H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. Because dividing cells dilute the label after each cell division, frequently dividing cells will lose the label over time while slow cycling cells will retain the label and thus are termed label retaining cells (LRCs). [H]TdR can be visualized by autoradiography and BrdU can be detected by immunofluorescence with anti-BrdU antibodies. Alternatively, a well-established tet-regulatable transgenic mouse model can be used to express histone H2B-GFP in epithelial proliferative cells and their dilution and retention of the GFP signal can be followed. In this chapter, we detail the steps to perform BrdU pulse-chase and H2B-GFP pulse-chase experiments to identify quiescent cells in the hair follicle.
Topics: Animals; Bromodeoxyuridine; Cell Differentiation; Cell Proliferation; Cells, Cultured; Hair Follicle; Mice; Mice, Transgenic; Resting Phase, Cell Cycle; Stem Cells
PubMed: 29030818
DOI: 10.1007/978-1-4939-7371-2_10 -
Cells Oct 2022Adult hippocampal neurogenesis is prone to modulation by several intrinsic and extrinsic factors. The anterior nucleus (AN) of the thalamus has extensive connections...
Adult hippocampal neurogenesis is prone to modulation by several intrinsic and extrinsic factors. The anterior nucleus (AN) of the thalamus has extensive connections with the hippocampus, and stimulation of this region may play a role in altering neurogenesis. We have previously shown that electrical stimulation of the AN can substantially boost hippocampal neurogenesis in adult rats. Here, we performed selective unilateral chemical excitation of the cell bodies of the AN as it offers a more specific and sustained stimulation when compared to electrical stimulation. Our aim is to investigate the long-term effects of KA stimulation of the AN on baseline hippocampal proliferation of neural stem cells and neurogenesis. Continuous micro-perfusion of very low doses of kainic acid (KA) was administered into the right AN for seven days. Afterwards, adult male rats received 5'-bromo-2'-deoxyuridine (BrdU) injections (200 mg/kg, i.p) and were euthanized at either one week or four weeks post micro-perfusion. Open field and Y-maze tests were performed before euthanasia. The KA stimulation of the AN evoked sustained hippocampal neurogenesis that was associated with improved spatial memory in the Y-maze test. Administering dexamethasone prior to and simultaneously with the KA stimulation decreased both the hippocampal neurogenesis and the improved spatial recognition memory previously seen in the Y-maze test. These results suggest that hippocampal neurogenesis may be a downstream effect of stimulation in general, and of excitation of the cell bodies of the AN in particular, and that stimulation of that area improves spatial memory in rats.
Topics: Male; Rats; Animals; Kainic Acid; Neurogenesis; Hippocampus; Neurons; Spatial Memory; Bromodeoxyuridine
PubMed: 36359809
DOI: 10.3390/cells11213413 -
Molecular and Cellular Biochemistry Sep 1976The mammalian epidermis is organized into layers of structurally different cells--the basal, spinous, granular and cornified layers--which represent steps in the... (Review)
Review
The mammalian epidermis is organized into layers of structurally different cells--the basal, spinous, granular and cornified layers--which represent steps in the differentiative process that terminates in cornification and desquamation. Investigation of the molecular mechanisms that control this ordered sequence of events provides clues to the etiology of certain epidermal pathologies. DNA synthesis and mitosis are normally restricted to the basal layer. Several substances have been implicated in the mitotic control of epidermal cells, the loss of mitotic activity being the first major step in normal keratinization. Investigations performed in this laboratory indicate that isolated differentiated nuclei can replicate their DNA which they are inhibited from doing in situ. Addition of a high speed supernate from homogenized differentiated cells inhibited this synthetic activity in vitro suggesting the existence of a cytoplasmic inhibitor of DNA synthesis. It is not known whether mitotic inhibition in differentiated epidermal cells is a function of the inhibition of DNA replication. Contrary to previous assumptions, recent experimental evidence clearly indicates that, unlike DNA synthesis, RNA synthesis occurs in differentiated cells. Correlated with this synthetic activity is the observation that a protein rich in histidine is specifically formed in the granular cells. This protein appears to be a component of the keratohyalin granules which fill the cells of the granular layer. Investigations were conducted in this laboratory to determine whether control of the synthesis of this protein occurs at the level of translation or transcription. Translation, in vitro, of mRNA obtained from isolated populations of each epidermal cell type suggested that control of protein synthesis in the differentiating epidermis is transcriptional, i.e. only in the granular cell is there an mRNA for the histidine-rich protein. Transcription, in vitro, of chormatin isolated from the separated cell populations produced RNA with a ratio of cytidine to uracil consistent with the predicted mRNA for this protein thus providing additional support for the hypothesis that epidermal differentiation is controlled at the level of 'gene-readout'.
Topics: Animals; Bromodeoxyuridine; Cell Differentiation; Cell Nucleus; DNA Replication; Mitosis; Rats; Skin; Skin Physiological Phenomena; Thymidine
PubMed: 790161
DOI: 10.1007/BF01741715 -
Nature Protocols May 2018This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content...
This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.
Topics: Animals; Bromodeoxyuridine; Cell Division; Cell Line; Chromatin Immunoprecipitation; DNA; DNA Replication; High-Throughput Nucleotide Sequencing; Mice; Mouse Embryonic Stem Cells; Staining and Labeling; Time
PubMed: 29599440
DOI: 10.1038/nprot.2017.148 -
Journal of Radiation Research Jun 2001Sensitization by bromodeoxyuridine (BrdUrd) and hyperthermia (HT) on cell reproductive death induced by ionizing radiation was analyzed using the linear-quadratic...
Sensitization by bromodeoxyuridine (BrdUrd) and hyperthermia (HT) on cell reproductive death induced by ionizing radiation was analyzed using the linear-quadratic [S(D)/S(0)=exp(-(alphaD + betaD2)]] model. Plateau-phase human lung tumor cells (SW-1573) and human colorectal carcinonoma cells (RKO) were treated with BrdUrd, radiation and HT. LQ-analysis was performed at iso-incubation dose and at iso-incorporation level of BrdUrd. and at iso-HT doses and iso-survival levels after HT. Clonogenic assays were performed 24 h after treatment to allow repair of potentially lethal damage (PLD). In SW cells BrdUrd. HT or the combination significantly increased the alpha-parameter (factor 2.0-5.7), without altering the beta-parameter. In RKO cells sensitization with BrdUrd increased both a (factor 1.4) and beta (factor 1.3) while HT only influenced beta (factor 2.1-4.0). The combination did not further increase the a and beta. The results indicate that BrdUrd has its main effect on the parameter alpha, dominant at clinically relevant radiation doses but that HT can affect both a and beta. The addition of BrdUrd and HT provides a method to enhance the efficacy of radiotherapy.
Topics: Bromodeoxyuridine; Cell Death; Hot Temperature; Humans; Radiation Tolerance; Radiation-Sensitizing Agents; Tumor Cells, Cultured
PubMed: 11599884
DOI: 10.1269/jrr.42.179