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AIM:To explore the mechanism of ATP synthase mitochondrial F0 complex H+ transporting,sub-unit F6(ATP5J)in affecting the metastasis of hepatoma carcinoma cells by regulating mitochondrial function-mediated cy-toskeletal remodeling.METHODS:Hepatocellular carcinoma cells Li-7 were used to construct the ATP5J overexpression and knockdown models.JC-1 staining was used to detect the mitochondrial membrane potential in each group,reactive oxygen species(ROS)levels were examined by DCHF-DA,and mitochondrial ATP fluorescence probe was used to assess mito-chondrial function.Cytoskeletal remodeling was detected with a microfilament green fluorescent probe(Actin-Tracker Green-488).Transwell assay was used to assess cell invasion ability.The expression levels of ATP5J and translocase of outer mitochondrial membrane 20(TOMM20)were determined by Western blot.RESULTS:Overexpression of ATP5J up-regulated mitochondrial membrane potential and mitochondrial ATP fluorescence intensity,induced cytoskeletal re-modeling,promoted cell invasion and TOMM20 expression,and inhibited ROS production(P<0.01).On the contrary,knockdown of ATP5J significantly decreased mitochondrial membrane potential and mitochondrial ATP fluorescence inten-sity,significantly decreased cell invasion ability and TOMM20 expression,promoted ROS production and blocked cyto-skeletal remodeling(P<0.01).CONCLUSION:ATP5J regulates mitochondrial energy transformation in hepatocellular carcinoma cells,and affects metastasis of hepatoma carcinoma cells by regulating mitochondrial membrane potential and mitochondrial ATP production-mediated cytoskeletal remodeling through TOMM20.
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Liver fibrosis is a dynamic wound-healing response characterized by the agglutination of the extracellular matrix (ECM). Si-Wu-Tang (SWT), a traditional Chinese medicine (TCM) formula, is known for treating gynecological diseases and liver fibrosis. Our previous studies demonstrated that long non-coding RNA H19 (H19) was markedly upregulated in fibrotic livers while its deficiency markedly reversed fibrogenesis. However, the mechanisms by which SWT influences H19 remain unclear. Thus, we established a bile duct ligation (BDL)-induced liver fibrosis model to evaluate the hepatoprotective effects of SWT on various cells in the liver. Our results showed that SWT markedly improved ECM deposition and bile duct reactions in the liver. Notably, SWT relieved liver fibrosis by regulating the transcription of genes involved in the cytoskeleton remodeling, primarily in hepatic stellate cells (HSCs), and influencing cytoskeleton-related angiogenesis and hepatocellular injury. This modulation collectively led to reduced ECM deposition. Through extensive bioinformatics analyses, we determined that H19 acted as a miRNA sponge and mainly inhibited miR-200, miR-211, and let7b, thereby regulating the above cellular regulatory pathways. Meanwhile, SWT reversed H19-related miRNAs and signaling pathways, diminishing ECM deposition and liver fibrosis. However, these protective effects of SWT were diminished with the overexpression of H19 in vivo. In conclusion, our study elucidates the underlying mechanisms of SWT from the perspective of H19-related signal networks and proposes a potential SWT-based therapeutic strategy for the treatment of liver fibrosis.
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Humans , RNA, Long Noncoding/genetics , Liver Cirrhosis/genetics , Liver/metabolism , Hepatic Stellate Cells/pathology , MicroRNAs/metabolism , Extracellular Matrix/metabolism , Drugs, Chinese HerbalABSTRACT
Abstract Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. Methodology Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. Results Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 μM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. Conclusion Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.
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Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.
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The ABRACL protein, the regulator of actin and cell motility, belongs to the HSPC280 family, and its conserved hydrophobic groove can interact with other proteins to facilitate actin motility and cellular activity. ABRACL is upregulated in tumor tissues and is closely linked with the proliferation and migration of tumor cells. A deeper understanding of the role of ABRACL in tumorigenesis and development may provide new ideas and insights for ABRACL to prevent or reverse tumor progression.
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Cell mechanics is essential to cell development and function,and its dynamics evolution reflects the physiological state of cells.Here,we investigate the dynamical mechanical properties of single cells under various drug conditions,and present two mathematical approaches to quantitatively character-izing the cell physiological state.It is demonstrated that the cellular mechanical properties upon the drug action increase over time and tend to saturate,and can be mathematically characterized by a linear time-invariant dynamical model.It is shown that the transition matrices of dynamical cell systems signifi-cantly improve the classification accuracies of the cells under different drug actions.Furthermore,it is revealed that there exists a positive linear correlation between the cytoskeleton density and the cellular mechanical properties,and the physiological state of a cell in terms of its cytoskeleton density can be predicted from its mechanical properties by a linear regression model.This study builds a relationship between the cellular mechanical properties and the cellular physiological state,adding information for evaluating drug efficacy.
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Intervertebral disc degeneration is the most common cause of chronic low back pain and the leading cause of disability in adults. The fact that lacking of effective treatment methods often causes a serious economic and social burden. Intervertebral disc degeneration is the result of multifactorial factors. The prevalence of intervertebral disc degeneration increases drastically with age, what is more, mechanical trauma, genetic predisposition,lifestyle factors and certain metabolic disorders. At present, the main treatment methods both pharmacological and surgical interventions just aim at relieving symptoms and improving function, and can not fundamentally reverse the process of intervertebral disc degeneration, which not only bring inevitable side effects and high cost, but also the long-term curative effect is limited. In theory, biological therapy can not only reverse or delay the process of it, but also can maximize preservation and restore the normal physiological function of the disc, which has been the focus and hot spot areas of research in recent years. The methods of inhibiting inflammation, promote the proliferation and division of residual cells, stem cell transplantation, cell scaffolds and new biomaterials all provide new ideas and direction for the treatment of intervertebral disc degeneration. This paper makes a review of the research progress in related fields, in order to provide a valuable reference for the selection of intervertebral disc degeneration treatment options.
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Objective:To investigate the mechanism of Candida albicans Int1 in regulating septin organization. Methods:A series of full-length and truncated fragments of Int1 were constructed and fused with green fluorescent protein (GFP). The intracellular localization of the fusion proteins was observed under a fluorescence microscope. The region in Int1 that was required for bud neck localization was identified. Full-length and fragments of Int1 were overexpressed in the yeast Saccharomyces cerevisiae and the changes in cell growth, cell morphology and septin organization were investigated to determine the functional region in Int1 that mediated the interaction with septin. Moreover, the co-localization of the region and septin was analyzed. Results:The full-length Int1 consisted of 1 661 amino acid residues. A middle region of 209 amino acid residues, Int1-M4 (739-947 aa), that could be localized at the bud neck during both small and large bud periods was identified. Overexpression of Int1-M4 led to significant growth defects, elongated bud and disorganized septin. In the cells with elongated bud, Int1-M4 and septin with abnormal structures could be co-localized.Conclusions:Int1-M4 (739-947 aa), the middle region of Int1 containing 209 amino acid residues, mediated the bud neck localization and the interaction with septin, playing an important role in regulating septin organization.
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@#Septin is a highly conserved class of GTP-binding proteins found in eukaryotes other than higher plants.The different subtypes of Septin can form higher order structures such as filamentous or ring-like structures in the form of heterodimer to perform their functions,which can regulate physiological processes such as yeast budding and cell division,as well as participating in the defense response of the host cell.Abnormal expression or mutation of Septin is closely related to the occurrence and development of tumors and neurological diseases.This paper summarized the physiological functions of the Septin family,the effects of Septin on the occurrence and development of tumors and neurological diseases,and the role of Septin in the host immune response.
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ObjectiveTo investigate the mechanism of icariin in ameliorating efferocytosis dysfunction and inflammatory response of alveolar macrophages induced by cigarette smoke extract via the peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathway. MethodThe untreated rat alveolar macrophages (NR8383) were taken as the blank group. The NR8383 cells treated with 10% cigarette smoke extract were divided into model, low-, medium-, and high-dose (10, 20, 40 μmol·L-1) icariin, PPARγ inhibitor, and PPARγ inhibitor + low-, medium-, and high-dose icariin groups. Alamar blue colorimetry was employed to examine the proliferation and toxicity of icariin on NR8383 cells. The efferocytosis rate of NR8383 cells was detected by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor-alpha (TNF-α), transforming growth factor-β1 (TGF-β1), and milk fat globule-epidermal growth factor 8 (MFG-E8). Western blot and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) were employed to determine the protein and mRNA levels, respectively, of PPARγ, CD36, and RAS-related C3 botulinum toxin substrate 1 (Rac1). ResultThe efferocytosis dysfunction model of NR8383 was established with the cigarette smoke extract. Compared with the blank control group, the model group showed decreased efferocytosis rate (P<0.05), elevated TNF-α level (P<0.05), lowered TGF-β1 and MFG-E8 levels (P<0.01), and down-regulated mRNA and protein levels of PPARγ, CD36, and Rac1 (P<0.05, P<0.01). Compared with the model group, the treatment with icariin increased the efferocytosis rate (P<0.05, P<0.01), lowered the TNF-α level (P<0.01), elevated TGF-β1 and MFG-E8 levels (P<0.05), and up-regulated the protein and mRNA levels of PPARγ, CD36, and Rac1 (P<0.05, P<0.01). Compared with icariin alone, PPARγ inhibitor + icariin decreased the efferocytosis rate (P<0.05) and down-regulated the protein and mRNA levels of PPARγ (P<0.05, P<0.01). In addition, PPARγ inhibitor + low-dose icariin down-regulated the protein level of CD36 (P<0.01) and PPARγ inhibitor + low-/medium-dose icariin up-regulated the protein level of Rac1 (P<0.05). ConclusionIcariin ameliorates the cigarette smoke extract-induced efferocytosis dysfunction of alveolar macrophage by regulating the PPARγ signaling pathway and cytoskeletal structure rearrangement.
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Objective @#To investigate the effects of aflatoxin B1 (AFB1) on the biophysical properties and cytoskeleton structure of human hepatocellular carcinoma cells (HCCs) . @*Methods@#HepG2 cells were respectively treated with 0 , 0. 01 , 0. 1 , 1 , 5 , 10 μmol/L AFB1 for 24 h and 48 h , and the cell viability was measured by CCK⁃8 kit.Based on this result , the influences of 10 μmol/L AFB1 on the osmotic fragility , membrane fluidity , electrophoretic mobility (EPM) and F ⁃actin structure of cells were analyzed. Subsequently , total RNAs were extracted and the PCR. @*Results@#The increased viability of HepG2 cells was induced by AFB1 in a dose⁃dependent manner after 48h treatment. After treated with 10 μmol/L AFB1 , the anti⁃hypotonic ability and EPM of HepG2 cells were en⁃hanced. The content of F ⁃actin in HepG2 cells increased obviously , while the mRNA expression levels of the main cytoskeleton binding proteins were altered. @*Conclusion @#AFB1 can affect the biophysical properties , cytoskeleton structure and its binding proteins of HepG2 cells , which may be directly related to its toxic action.
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Abstract Background As a progressive cerebrovascular disease, Moyamoya Disease (MMD) is a common cause of stroke in children and adults. However, the early biomarkers and pathogenesis of MMD remain poorly understood. Methods and material This study was conducted using plasma exosome samples from MMD patients. Next-generation high-throughput sequencing, real-time quantitative PCR, gene ontology analysis, and Kyoto Encyclopaedia of Genes and Genomes pathway analysis of ideal exosomal miRNAs that could be used as potential biomarkers of MMD were performed. The area under the Receiver Operating Characteristic (ROC) curve was used to evaluate the sensitivity and specificity of biomarkers for predicting events. Results Exosomes were successfully isolated and miRNA-sequence analysis yielded 1,002 differentially expressed miRNAs. Functional analysis revealed that they were mainly enriched in axon guidance, regulation of the actin cytoskeleton and the MAPK signaling pathway. Furthermore, 10 miRNAs (miR-1306-5p, miR-196b-5p, miR-19a-3p, miR-22-3p, miR-320b, miR-34a-5p, miR-485-3p, miR-489-3p, miR-501-3p, and miR-487-3p) were found to be associated with the most sensitive and specific pathways for MMD prediction. Conclusions Several plasma secretory miRNAs closely related to the development of MMD have been identified, which can be used as biomarkers of MMD and contribute to differentiating MMD from non-MMD patients before digital subtraction angiography.
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A well-coordinated process is required to construct a complicated structure like the cell wall, which consists of several elements that must be joined appropriately from various sources inside the cell. In order to successfully moderate dynamic responses to developmental and environmental signals, further complexity is necessary. The plasma membrane is continually and actively transporting sugars, enzymes, and other cell wall elements throughout diffused development. Actin filaments and microtubules make up the cytoskeletal pathways used to transport cell wall elements in vesicles during cell division. In addition to these elements, other proteins, vesicles and lipids are transported from and to the cell plate while cytokinesis occurs. Adding additional cell wall material or building a new cell wall requires a rearrangement of the cytoskeleton, which we examine in this review first. We next look at the commonalities between these two processes. Our next topic is the transport of cell wall-building polysaccharides and enzymes via motor proteins and other interactions with the cytoskeleton. Final thoughts on cytokinesis-generated cell walls include a look at some of their unique properties.
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Objective From the perspective of biophysics and immunology, the effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on biophysical characteristics, cytoskeleton and migration ability of mouse derived dendritic cells (DCs) were analyzed, so as to explore the effect of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) on immune function of DCs and its potential mechanism. Methods The bone marrow-derived monocytes from C57BL/6J mice were isolated and induced to differentiate into immature dendritic cells (imDCs) by 20 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4), After 6 days, 100 ng/mL lipopolysaccharide was added to induce mature dendritic cells (mDCs). Further the morphological observation and positive rate of CD11c in imDCs and mDCs were analyzed, Then at different concentrations of EPA and DHA (0-60 μmol/L), the cell viability and apoptosis of DCs were detected by cell counting kit-8 (CCK-8) kit and flow cytometry. After the optimal concentration of EPA and DHA were determined, the changes of the membrane fluidity, electrophoretic mobility (EPM) and osmotic fragility of DCs were separately detected by the fluorescence polarization, cell electrophoresis and concentration gradient. The expression of filamentous actin (F-actin) was detected by the immunofluorescence. Finally, the migration ability of DCs was detected by the Transwell system. Results The positive rate of CD11c in DCs was about 80%. The viability of DCs decreased in a dose-dependent manner under the action of EPA and DHA of different concentrations, which didn’t induce the apoptosis of DCs. Under the action of 50 μmol/L EPA and DHA, the osmotic fragility and EPM of DCs significantly decreased, and the membrane fluidity significantly increased. The expression of F-actin in DCs obviously increased, and the migration rate of cells obviously decreased. Conclusions ω-3 PUFAs may affect the cytoskeleton structure and biophysical characteristics of DCs, inhibit the migration, and then affect its immune function.
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Objective:To investigate the protective role of Yes-associated protein(YAP)in intestinal epithelial barrier injury.Methods:The intestinal epithelial barrier model was established by culturing human colorectal adenocarcinoma cell line Caco-2 cells, which were divided into four groups: control group, Caco-2 monolayers did not receive any treatment; recombinant human tumor necrosis factor-α(rhTNF-α)group, 100 μg/L of rhTNF-α was added to Caco-2 monolayers; vector+ rhTNF-α group, Caco-2 monolayers were first added with control plasmid pcDNA3.1-vector, and 100 μg/L rhTNF-α was added 24 hours later; YAP+ rhTNF-α group, Caco-2 cells with barrier construction were first added with pcDNA3.1-YAP, and 100 μg/L rhTNF-α was added 24 hours later.Realtime-PCR and Western blot were used to evaluate YAP mRNA and protein expression level.Epithelial permeability was assayed by trans-epithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran 40(FD-40 flu). Cellular distribution of F-actin was assayed by immunofluorescence staining.Results:Compared with control group[(607.3±29.3)Ω·cm 2], TEER of rhTNF-α group[(265.3±32.7)Ω·cm 2] decreased, while TEER of YAP+ rhTNF-α group[(387.0±18.7)Ω·cm 2]increased compared with rhTNF-α group, the differences were statistically significant( P<0.001). The FD-40 flux of rhTNF-α(22.7%±0.5%) group was higher than that of the control group(6.3%±0.9%), while the FD-40 flux of Yap + rhTNF-α group(12.2%±0.8%) was lower than that of rhTNF-α group, the differences were statistically significant( P<0.001). Immunofluorescence staining showed that compared with the control group, the cytoskeletal F-actin fiber dense spot decreased in rhTNF-α group, and some cells showed obvious trans-cellular stress fiber structure, while the peripheral actin band was clear in YAP+ rhTNF-α group, and the intracellular stress fiber decreased.YAP+ TNF-α group appeared as a clear, peripheral actin ribbon with a decrease in cytoplasmic stress fibres. Conclusion:YAP overexpression significantly inhibits TNF-α induced decline of TEER, and increases of FD-40 flux and F-actin rearrangement of Caco-2.YAP could ameliorate TNF-α induced intestinal epithelial barrier injury by regulating cytoskeleton F-actin.
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Objective To story the effect of long non-coding RNA (IncRNA) cytoskeleton regulatory RNA (CYTOR) targeting microRNA (miR)-1246 on cell damage in Parkinson' s disease (PD) models. Methods SK-N-SH cells were exposed to 100 |xmol/L 1 -methyl-4-phenylpyridinium (MPP
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BACKGROUND Profilin proteins (PRFs) are small (1215 kD) actin-binding protein, which play a significant role in cytoskeleton dynamics and plant development via regulating actin polymerization. Profilins have been well documented in Arabidopsis, Zea mays L. as well as Phaseolus vulgaris, however no such fully characterization of rice (Oryza sativa L.) profilin gene family has been reported thus far. RESULTS In the present study, a comprehensive genome-wide analysis of rice PRF genes was completed and three members were identified. OsPRF1 and OsPRF2 shared 98.5% similarity (6 nucleotide divergence), but the deduced amino acid sequences of OsPRF1 and OsPRF2 are fully identical. In contrast, the OsPRF3 presents relatively lower similarity with OsPRF1 and OsPRF2. Phylogenetic analysis also support that OsPRF1 has a closer relationship with OsPRF2. Expression pattern analysis revealed the differential expression of OsPRFs in tissues of mature plant, which suggested the potential spatial functional specificity for rice profilin genes. Subcellular localization analysis revealed the OsPRFs were localized in cytoplasm and nucleus and all of them could bind actin monomers. Furthermore, abiotic stresses and hormones treatments assay indicated that the three OsPRF genes could be differentially regulated, suggesting that OsPRF genes might participate in different stress processes in rice. CONCLUSIONS Taken together, our study provides a comprehensive analysis of the OsPRF gene family and will provide a basis for further studies on their roles in rice development and in response to abiotic stresses
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Plant Proteins/genetics , Oryza/genetics , Genome, Plant , Profilins/geneticsABSTRACT
SUMMARY: Cellular differentiation is a highly regulated process that has vast implications for the mechanics of the cell. The interplay between differentiation induced cytoskeletal mechanical changes and strain on the nucleus is a potential cause of gene level changes. This study explores mechanical changes in SH-SY5Y neural cells during differentiation mediated by Retinoic Acid (RA) across Days 0 through 9. Findings suggest that cellular elongation increases 1.92-fold over a 10-day differentiation period, from 48.97 ±16.85µm to 93.96 ± 31.20 µm over 3 repeated trials and across multiple cells analyzed on ImageJ. Nuclear elongation increases less substantially from 17.51 ± 2.71 µm to 23.26 ± 3.10 µm over 3 repeated trials and across multiple cells. Results are statistically significant at a significance level of α = 0.05. This study is one of the first studies to show that during the process of RA mediated neural differentiation in SH-SY5Y neural cells, nuclear elongation is initially not significantly correlated with cellular elongation, but it becomes correlated during the differentiation process with an overall correlation coefficient of 0.4498 at a significance level of α = 0.05. Given the time course of the mechanical changes and the known coupling between the cytoskeleton and nuclear lamina, this study suggests a causative and correlative relationship between neurite-driven cellular elongation and nuclear elongation during neural differentiation.
RESUMEN: La diferenciación celular es un proceso altamente regulado que tiene vastas implicaciones para la mecánica de la célula. La interacción entre los cambios mecánicos citoesqueléticos inducidos por la diferenciación y la tensión en el núcleo es una causa potencial de cambios a nivel genético. Este estudio explora los cambios mecánicos en las células neurales SH-SY5Y durante la diferenciación mediada por el ácido retinoico (RA) durante los días 0 a 9. Los resultados sugieren que el alargamiento celular aumenta 1,92 veces durante un período de diferenciación de 10 días, de 48,97 ± 16,85 µm a 93,96 ± 31,20 µm en 3 ensayos repetidos y en múltiples células analizadas en Image J. El alargamiento nuclear aumenta menos sustancialmente de 17,51 ± 2,71 µm a 23,26 ± 3,10 µm durante 3 ensayos repetidos y en múltiples células. Los resultados son estadísticamente significativos a un nivel de significancia de α = 0,05. Esta investigación es uno de los primeros estudios en demostrar que durante el proceso de diferenciación neural mediada por RA en las células neurales SH-SY5Y, el alargamiento nuclear inicialmente no se correlaciona significativamente con el alargamiento celular, pero se correlaciona durante el proceso de diferenciación con un coeficiente de correlación global de 0,4498 a un nivel de significancia de α = 0,05. Dado el curso temporal de los cambios mecánicos y el acoplamiento conocido entre el citoesqueleto y la lámina nuclear, este estudio sugiere una relación causal y correlativa entre el alargamiento celular impulsado por neuritas y el alargamiento nuclear durante la diferenciación neural.
Subject(s)
Cytoskeleton , Cell Differentiation , Cell Nucleus , NeuronsABSTRACT
Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.
Subject(s)
Cytoskeletal Proteins , Endothelial Cells , Extracellular Traps , Integrins , Intercellular Adhesion Molecule-1 , Lipopolysaccharides/pharmacology , Macrophages , NeutrophilsABSTRACT
Objetivo: identificar, describir y diferenciar las características fenotípicas de los fibroblastos gingivales (FGs) en pacientes con hiperplasia gingival idiopática (HGI) e individuos periodontalmente sanos. Métodos: los FGs fueron aislados a partir de tejido gingival de individuos periodontalmente sanos (n=2) y pacientes con HGI (n=2). Los FGs se cultivaron en el medio DMEM (Dulbecco's Modified of Eagle Medium) a 37°C con 5% de CO2. La identificación y localización de la actina, vimentina y mitocondrias en FGs fue realizada y evaluada microscópicamente mediante inmunofluorescencia con anticuerpos monoclonales. La capacidad de migración de los FGs en los pacientes con HGI e individuos sanos también fue estudiada. Resultados: todos los FGs fueron mononucleares, fusiformes y con prolongaciones citoplasmáticas visibles. La faloidina permitió identificar una densa red de actina en los FGs de pacientes con HGI, contrariamente a los FGs de individuos periodontalmente sanos. La vimentina y mitocondrias fueron identificadas en los FGs de individuos sanos y pacientes con HGI sin ninguna alteración en su expresión y localización. La migración de la monocapa de los FGs indicó una actividad de migración celular importante en los FGs de los pacientes con HGI, en relación a los FGs de los individuos periodontalmente sanos. Conclusión: los FGs de pacientes con HGI conservan características fenotípicas celulares similares a los FGs de individuos periodontalmente sanos. Sin embargo, los FGs de pacientes con HGI simulan tener una mayor capacidad migratoria que amerita ser explorada en futuros trabajos de investigación.
Objective: To identify and to describe the phenotypic characteristics of gingival fibroblasts from patients with idiopathic gingival hyperplasia (IGH) and periodontally healthy individuals. Methods: Gingival fibroblasts (GFs) were isolated from gingival tissue from periodontally healthy individuals (n=2) and patients with IGH (n=2). The GFs were grown in DMEM (Dulbecco's Modified of Eagle Medium) at 37°C with 5% CO2. The identification and location of actin, vimentin and mitochondria in GFs were performed and evaluated microscopically by immunofluorescence with monoclonal antibodies. The migration capacity of GFs from IGH and healthy individuals was also studied. Results: All the GFs were mononuclear, fusiform and with visible cytoplasmic extensions. The phalloidin allowed to identify a dense actin network in the GFs of patients with IGH, contrary to the GFs of periodontally healthy individuals. Vimentin and mitochondria were identified in the GFs of healthy individuals and patients with IGH without any alteration in their expression and location. Monolayer migration of GFs indicates significant cell migration activity in the GFs of patients with IGH in relation to the GFs of periodontally healthy individuals. Conclusion: GFs from patients with IGH retain cellular phenotypic characteristic similar to GFs from periodontally healthy individuals. However, the GFs of patients with IGH simulate having a greater migratory capacity that deserves to be explored in future research works.