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Molecular Biology of the Cell Jul 2020The dynamic rearrangement of the actin cytoskeleton is an essential component of many mechanotransduction and cellular force generation pathways. Here we use periodic...
The dynamic rearrangement of the actin cytoskeleton is an essential component of many mechanotransduction and cellular force generation pathways. Here we use periodic surface topographies with feature sizes comparable to those of in vivo collagen fibers to measure and compare actin dynamics for two representative cell types that have markedly different migratory modes and physiological purposes: slowly migrating epithelial MCF10A cells and polarizing, fast-migrating, neutrophil-like HL60 cells. Both cell types exhibit reproducible guidance of actin waves (esotaxis) on these topographies, enabling quantitative comparisons of actin dynamics. We adapt a computer-vision algorithm, optical flow, to measure the directions of actin waves at the submicron scale. Clustering the optical flow into regions that move in similar directions enables micron-scale measurements of actin-wave speed and direction. Although the speed and morphology of actin waves differ between MCF10A and HL60 cells, the underlying actin guidance by nanotopography is similar in both cell types at the micron and submicron scales.
Topics: Actin Cytoskeleton; Actins; Cell Movement; Cytoskeleton; Epithelial Cells; HL-60 Cells; Humans; Image Processing, Computer-Assisted; Mechanical Phenomena; Mechanotransduction, Cellular; Models, Biological
PubMed: 32023172
DOI: 10.1091/mbc.E19-11-0614 -
Scientific Reports Nov 2021Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk...
Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.
Topics: Aged; Case-Control Studies; Endothelium, Vascular; Extracellular Traps; Female; HL-60 Cells; Humans; Intercellular Adhesion Molecule-1; Male; Renal Dialysis; Renal Insufficiency, Chronic; Uremia; Vascular Diseases
PubMed: 34728714
DOI: 10.1038/s41598-021-00863-w -
International Journal of Molecular... Sep 2021Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve...
Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.
Topics: Acetophenones; Cell Differentiation; Cell Proliferation; Cell Shape; Cell Survival; Electron Spin Resonance Spectroscopy; Free Radicals; HL-60 Cells; Humans; Hydroxyl Radical; Monocytes; NADP; Proteins; Staining and Labeling; Tetradecanoylphorbol Acetate; U937 Cells
PubMed: 34576127
DOI: 10.3390/ijms22189963 -
MAbs 2020Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular...
Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular traps (NETs), are involved in multiple pathological processes, and NET formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity toward intact NS but not to individual NS components, indicating that 2C5 could potentially target NS in NETs. In this study, NETs were generated using neutrophils and HL-60 cells differentiated into granulocyte-like cells. The specificity of 2C5 toward NETs was evaluated by ELISA, which showed that it binds to NETs with the specificity similar to that for purified nucleohistone substrate. Immunofluorescence showed that 2C5 stains NETs in both static and perfused microfluidic cell cultures, even after NET compaction. Modification of liposomes with 2C5 dramatically enhanced liposome association with NETs. Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies.
Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Extracellular Traps; HL-60 Cells; Humans; Mice; Mice, Inbred BALB C
PubMed: 33323006
DOI: 10.1080/19420862.2020.1850394 -
International Journal of Medical... 2021Although accumulating evidence has revealed that metallothioneins (MTs) and its family member MT2A are strongly linked to the risk of various solid tumors, researches on...
Although accumulating evidence has revealed that metallothioneins (MTs) and its family member MT2A are strongly linked to the risk of various solid tumors, researches on the occurrence and development of acute myeloid leukemia (AML) have rarely been investigated. Here, we constructed a lentiviral vector with MT2A over-expression and the interfering plasmids with MT2A expression inhibition to study the influence of MT2A on the bioactivities of HL60 cells. After cells were infected with a lentiviral vector containing the MT2A gene, both transcription and translation levels of MT2A were significantly increased in the over-expressed group in comparison with control groups. In vitro experiments, all results demonstrated that cell reproductive capacity was inhibited, but cell apoptosis rate was significantly increased. Together, the expression of apoptosis-related protein Bcl2 was remarkably reduced, while a high expression level of Bax protein was detected. Further experiments revealed that up-regulation of MT2A induced cell apoptosis and promoted G2/M phase arrest. The mechanism may be associated with down-regulated p-IκB-α and cyclinD1 expression and up-regulated IκB-α expression in the nuclear factor-kappaB (NF-κB) pathway. On the contrary, MT2A expression was down-regulated by interfering plasmids. We found that cell proliferative potential was notably increased in the interfering group compared with the negative and untreated group. What's more, MT2A may be closely related to AML cell proliferation and function via the NF-κB signal pathway.
Topics: Apoptosis; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Metallothionein; NF-KappaB Inhibitor alpha; Signal Transduction; Up-Regulation
PubMed: 34220318
DOI: 10.7150/ijms.57821 -
Cell Death & Disease Feb 2021MYB plays vital roles in regulating proliferation and differentiation of hematopoietic progenitor cells, dysregulation of MYB has been implicated in the pathogenesis of...
MYB plays vital roles in regulating proliferation and differentiation of hematopoietic progenitor cells, dysregulation of MYB has been implicated in the pathogenesis of leukemia. Although the transcription of MYB has been well studied, its detailed underlying regulatory mechanisms still remain elusive. Here, we detected the long-range interaction between the upstream regions, -34k and -88k, and the MYB promoter in K562, U937, and HL-60 cells using circularized chromosome conformation capture (4C) assay, which declined when MYB was downregulated during chemical-induced differentiation. The enrichment of enhancer markers, H3K4me1 and H3K27ac, and enhancer activity at the -34k and -88k regions were confirmed by ChIP-qPCR and luciferase assay respectively. ChIP-qPCR showed the dynamic binding of GATA1, TAL1, and CCAAT/enhancer-binding protein (C/EBPβ) at -34k and -88k during differentiation of K562 cells. Epigenome editing by a CRISPR-Cas9-based method showed that H3K27ac at -34k enhanced TF binding and MYB expression, while DNA methylation inhibited MYB expression. Taken together, our data revealed that enhancer elements at -34k are required for MYB expression, TF binding, and epigenetic modification are closely involved in this process in human myeloid leukemia cells.
Topics: Binding Sites; CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; DNA Methylation; Enhancer Elements, Genetic; Epigenesis, Genetic; GATA1 Transcription Factor; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myb; T-Cell Acute Lymphocytic Leukemia Protein 1; U937 Cells
PubMed: 33637692
DOI: 10.1038/s41419-021-03515-z -
Molecules (Basel, Switzerland) Jun 2021A phytochemical investigation of the leaves of the medicinal plant led to the isolation of the two new degraded abietane lactone diterpenoids rubesanolides F () and G...
A phytochemical investigation of the leaves of the medicinal plant led to the isolation of the two new degraded abietane lactone diterpenoids rubesanolides F () and G (). Their structures were elucidated based on the analyses of the HRESIMS and 1D/2D NMR spectral data, and their absolute configurations were determined by ECD spectrum calculations and X-ray single crystal diffraction methods. Compounds and , with a unique γ-lactone subgroup between C-8 and C-20, were found to form a carbonyl carbon at C-13 by removal of the isopropyl group in an abietane diterpene skeleton. Rubesanolide G () is a rare case of abietane that possesses a -fused configuration between rings B and C. The two isolates were evaluated for their biological activities against two cancer cell lines (A549 and HL60), three fungal strains (, and ) and three bacterial strains (, and ).
Topics: A549 Cells; Abietanes; Anti-Infective Agents; Antineoplastic Agents, Phytogenic; Bacteria; Fungi; HL-60 Cells; Humans; Isodon; Lactones; Neoplasms; Plant Leaves
PubMed: 34202760
DOI: 10.3390/molecules26133865 -
European Journal of Cell Biology Jun 2024We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid...
We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed "cloud"-like NETs, whereas those with low or no gelsolin formed long "filamentous" NETs.
Topics: Humans; Extracellular Traps; Neutrophils; Actin Cytoskeleton; HL-60 Cells; Actins; Gelsolin
PubMed: 38555846
DOI: 10.1016/j.ejcb.2024.151407 -
Molecules (Basel, Switzerland) Jun 2021(Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is...
(Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REO) versus leaf (REO) essential oils revealed significant differences. REO was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REO was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REO and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with -formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca mobilization and chemotaxis in human neutrophils and agonist-induced Ca mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca] desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.
Topics: Flowers; HL-60 Cells; Humans; Immunologic Factors; Immunomodulation; Monoterpenes; Neutrophils; Oils, Volatile; Plant Leaves; Receptors, Formyl Peptide; Rhododendron; Sesquiterpenes
PubMed: 34203809
DOI: 10.3390/molecules26123652 -
Life (Basel, Switzerland) May 2022In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the...
In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL.
PubMed: 35743820
DOI: 10.3390/life12060789