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CBI websites generally use certain cookies to enable better interactions with. own risk, without recourse to Applied Biosystems. 63 It could be possibly a cell-type dependent mechanism that differentially regulates an epigenetic switch in monocytes vs effector T lymphocytes. Applied Biosystems is not responsible for providing any end-user support.

Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2CT method. The DNA was then analyzed by qPCR to check the H3K27 acetylation level using Epitect qPCR primers ( Table S1 ) specific to the different regions close to the transcription start site of CCL2 proximal promotor region ( Figure S1 ). Herein, we present the data, suggesting that IFN-/LPS co-stimulation has the potential to augment inflammation via the increased CCL2 production by monocytic cells in metabolic condition, such as obesity. Vous navez pas de profil ?

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All p-values < 0.05 were considered significant (ns, non-significant, *p< 0.05, **p< 0.01, and ***p< 0.001). 1Immunology & Microbiology Department, Dasman Diabetes Institute, Kuwait City, Kuwait; 2Genetics & Bioinformatics, Dasman Diabetes Institute, Kuwait City, Kuwait; 3Animal and Imaging Core Facility, Dasman Diabetes Institute, Kuwait City, Kuwait; 4Institut Necker Enfants Malades (INEM), French Institute of Health and Medical Research (INSERM), Immunity & Metabolism of Diabetes (IMMEDIAB), Universit de Paris Cit, Paris, France Figure 1 IFN- and LPS cooperatively induce CCL2 in monocytic cells/macrophages. The extensive life sciences portfolio includes: With a wide selection of options within each category, youll find a product to fit your unique needs and budget. After washing thrice, substrate solution was added (100 L/well) and plates were again incubated at room temperature for 20 min in the dark.

This page is dedicated to featured products, resources, and valuable information pertaining to this rapidly developing area. LabX has a large variety of resources for the expanding world of materials science. ChIP was performed using the Simple ChIP Enzymatic Chromatin Immunoprecipitation Kit (Cell Signaling Technology, Inc), according to the manufacturers instructions. For acetylation, THP-1 monocytic cells in triplicate wells of 6-well plates (1x106 cells/mL per well) cultured in RPMI-1640 complete medium were treated with IFN- (10 ng/mL), LPS (10 ng/mL) alone, or in combination for 4 h, in a humidified incubator (5% CO2) and mock-treated cells served as experimental control. Applied Biosystems Participates in BioIT Alliance by Microsoft. Figure 2 Synergistic expression of CCL2 is dependent on STAT1. You can read more about your cookie choices at our privacy policyhere. These results show the synergistic effect of IFN-/LPS co-stimulation on the production of CCL2 by monocytic cells and macrophages. Briefly, cell lysates were resolved using 12% SDS-PAGE and blots were probed with rabbit anti-human H3K27 antibody (1:1000 dilution) at 4C overnight.

All p-values <0.05 were considered significant (ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). biosystems applied catalog

Chronic upregulation of CCL2 is often mediated by epigenetic mechanisms consisting of histone modification, and nucleosome positioning. The digested chromatin fragments were subjected to immunoprecipitation using primary antibodies specific to H3K27 (Cell Signaling Technology, Inc), Histone H3 (Positive IP control; Cell Signaling Technology, Inc.) and Normal Rabbit IgG (Negative IP control, Cell Signaling Technology, Inc), for overnight at 4C.

Similar results were obtained using primary human monocytes ( Figure 1C ). Proinflammatory M1-like polarization of macrophages requires a pro-oxidant Th1 microenvironment, marked by two initiation signals, such as IFN- and LPS or TNF-. 58 Whether sequentially or simultaneously treated with IFN- and LPS, STAT1 appears to be a convergence point for proinflammatory immune responses in monocytes or macrophages, 59 which is in agreement with the synergy between these two proinflammatory stimuli for CCL2 expression.

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Western blots show that IFN- treatment enhances the H3K27 acetylation in monocytic cells, whereas LPS treatment does not induce H3K27 acetylation ( Figure 3A and B ). including the software tools, is conditioned on your acceptance of the above terms, Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Moreover, we confirm that IFN- mediates stat1 phosphorylation in monocytic cells ( Figure 2D ). In this complex, Jak1/Jak2 transactivates each other and phosphorylate IFNGR1, forming a paired set of STAT1 docking sites on the ligand-activated IFN- receptor. Data (mean SEM, n = 3) were expressed as fold enrichment levels and were compared for different treatments against control using one-way ANOVA with post-hoc Tukeys test.

Interestingly, increased expression of CCL2 (C) mRNA (**p<0.01) and (D) secreted protein (***p<0.001) were observed in the cells that were treated with HDAC-inhibitor TSA before stimulation with LPS only as compared to similarly stimulated cells that were not pre-treated with TSA, suggesting that TSA priming could mimic effect of and substitute for IFN- in cooperativity with LPS. Inhibition or Activation of Acetylation by Pharmacological Agents Affects the Synergy Between IFN- and LPS Conclusion: Our findings suggest that IFN-, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

Device and method for making discrete volumes of a first fluid in contact with a second fluid, which are immiscible with each other, IFN- and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation, Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype, Impact of HOXB4 and PRDM16 Gene Expressions on Prognosis and Treatment Response in Acute Myeloid Leukemia Patients, Thermo Fisher Scientific Updates Real-Time PCR System to Comply with IVDR Regulations, Examining an Association of Single Nucleotide Polymorphisms with Hyperuricemia in Chinese Flight Attendants.

In this regard, we found that priming effect of IFN- followed by LPS stimulation was significantly more robust, than other way around, in driving CCL2 gene and protein expression in monocytic cells ( Figure 1F and G ) 28 , 48 Overall, the mechanisms underlying the IFN- priming effect appear to be complex, such as involving CD14 (LPS receptor) upregulation, 49 altered mRNA turnover, 50 inflammatory autocrine signaling, 28 and the role of CR3/CR4 complement receptors. (D) Western blot showing phosphorylation of STAT1 after IFN- (10 ng/mL) treatment over time indicates the optimal STAT1 phosphorylation at 120 min. Of note, H3K27 modification plays a key role in regulating gene expression, where acetylation (H3K27Ac) and methylation (H3K27me3) promote gene expression and repression/silencing, respectively. Applied Biosystems by Life Technologies, The SOLiD System: Next-Generation Sequencing, Applied Biosystems Participates in BioIT Alliance by Microsoft. Please do not call Applied Biosystems Customer/Technical Support. IFN- and LPS Synergistically Induce CCL2 in Monocytic Cells 810 It has been well documented that there is an increase in macrophage accumulation in white adipose tissue of obese mice and humans, and these macrophages predominantly contribute to induction of local and systemic inflammation and insulin resistance. 13 Chronic low-grade inflammation is a hallmark of obesity, resulting from continuous production of inflammatory cytokines and chemokines by the expanding adipose tissue under the state of overnutrition. Human monocytic leukemia THP-1 cell line was purchased from American Type Culture Collection (ATCC) and propagated in RPMI-1640 culture medium (Gibco, Life Technologies, Grand Island, USA), containing 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY, USA), 2 mM glutamine (Gibco, Invitrogen, Grand Island, NY, USA), 1 mM sodium pyruvate, 10 mM HEPES, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, Invitrogen, Grand Island, NY, USA). 40 Our data show that stimulation of monocytes/macrophages with IFN- and/or LPS induces CCL2 expression.

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To ensure that you see the most relevant information, please select your country. Chemokines are small chemoattractant proteins that impact the trafficking of immune cells to the sites of inflammation. Biosystems and we neither support nor make any warranty for such resources.

Veuillez vous connecter pour afficher les prix de votre compte et la disponibilit de vos produits. The data show the reduced expression of CCL2 (A) mRNA (***p<0.001) and (B) secreted protein (**p<0.01) in the cells that were treated with HAT-inhibitor anacardic acid before co-stimulation with IFN- and LPS as compared to similarly stimulated cells that were not pre-treated with anacardic acid. Macrophage Differentiation Applied Biosystems GeneAmp PCR System 2700, Bio-Rad Laboratories, Inc. MyCycler Personal Thermal Cycler, Esco Technologies Swift Maxi Thermal Cyclers, Life Technologies QuantStudio 3 Real-Time PCR System, Life Technologies QuantStudio 5 Real-Time PCR System, Applied Biosystems ABI ViiA 7 Real-Time 384 & 96 &.

mix master applied biosystems thermo scientific fisher biocompare write 53 Overall, increasing evidence supports that IFN- primes cells and synergizes with LPS to activate inflammatory programs via diverse molecular mechanisms, such as chromatin remodeling and metabolic reprogramming. Introduction

Adherent cells were then washed and incubated in serum-free RPMI media for further three days before they were considered ready for treatment. Shop now for new and used products for materials research and testing applications. Given that IFN- priming induces H3K27 acetylation, a histone mark for active enhancers and promoters of several genes, 34 we asked whether the H3K27 acetylation was involved in this synergistic upregulation of CCL2 gene expression by IFN-. limiting the foregoing, your use of such resources is subject to any applicable license or ChIP-qPCR analysis reveals that IFN- treatment promotes H3K27 acetylation on all CCL2 promoter regions ( Figure 3D and I ). Copyright 2022 CB Information Services, Inc. All rights reserved.

Browse ads and contact the seller directly or request a quote for more details. Figure 4 Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate synergy between IFN- and LPS for the CCL2 production. Future studies will be needed to identify the additional mechanisms of induction of inflammatory and repression of anti-inflammatory pathways by IFN-/LPS co-stimulation and to find out how they relate to monocyte/macrophage functions in the context of metabolic inflammation. 4311, Email [emailprotected] Peripheral blood mononuclear cells (PBMCs) were isolated using HistoPaque density gradient method as described. including the applicable license terms, if any, and your use of any of the resources herein

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The major STAT protein activated by IFN- is STAT1, and many IFN- functions are mediated through direct activation of immune effector processes by STAT1.

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Author Contributions Conclusion Now you can shop LabX to find antibodies, reagents, kits, and related tools for your research and clinical needs. RNA samples (1 g each) were reverse transcribed into cDNA by using random hexamer primers and TaqMan reverse transcription reagents (High-Capacity cDNA Reverse Transcription Kit; 4368814, Applied Biosystems, CA, USA). Comments regarding this site and Applied Biosystems resources provided herein LabX keeps pace with this industry by offering a wide range of instruments and solutions for the most demanding questions. 1115

55 , 56 We further show that the IFN-LPS synergy for CCL2 expression in monocytic cells was STAT1-dependent as the genetic knockdown of STAT1 led to a significant CCL2 suppression at the transcriptional and translational levels. should be submitted to software.community@appliedbiosystems.com. indicate your understanding and acceptance of these terms. Synergistic amplification of CCL2 expression by co-stimulation was a plausible outcome of this study. The exploding cannabis industry has created the need for high quality processing and testing products for laboratory operations of all scales.

THP-1 cells were differentiated into macrophages as previously described. CCL2 protein was measured in cell supernatants using commercial ELISA kit. Keywords: CCL2, monocytes/macrophages, LPS, IFN-, H3K27 ac, inflammation To confirm the role of histone acetylation, we examined whether the inhibition of histone acetyl transferases (HATs) impacted the IFN-/LPS synergy for CCL2 gene expression and protein secretion.

The data show significant suppression of STAT1 mRNA expression in cells transfected with STAT1 siRNA compared to control siRNA transfected cells (**p< 0.01). Ccl2 mRNA expression was determined by real-time RT-PCR and target gene expression were normalized to GAPDH expression. In particular, histone acetylation has been shown to play important roles in upregulating CCL2 expression in inflammatory cells. To further identify acetylation levels on the CCL2 promoter regions, monocytic cells were treated with IFN-, LPS, or IFN-+LPS for 24 hrs and cell lysates were prepared. CCL2 secreted protein expression was detected by ELISA as described in materials and methods. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2CT method. The LabX marketplace has all the laboratory products, equipment, and accessories for your cell culture needs. In partial agreement with this, a previous study showed that IFN- stimulation induced CCL2 expression in human astrocytoma cells. H3K27 acetylation levels induced by treatments compared to control (vehicle) were detected by qPCR using primers specific to the closest regions of transcription start site at the CCL2 promotor. For example, phagocytic and microbicidal activities, as well as expression of cytokines, chemokines, and antigen presenting molecules. Students t-test was used to determine if the means of the two data sets differed significantly from each other, and the one-way ANOVA followed by post-hoc Tukeys test were used to compare the means of more than two groups, with one categorical independent variable and one quantitative dependent variable. (A) THP-1 monocytic cells were transfected with control/scrambled siRNA or STAT1 siRNA and incubated for 40h. Integrity/quality of isolated RNA was checked before gene expression analysis by running the aliquots of RNA samples on a denaturing agarose gel stained with ethidium bromide, which yielded 2:1 ratio of the 28S and 18S rRNA bands, respectively (data not shown). Web resources for developers of life science software. The field of Bioprocessing technology is expanding, with advances in upstream and downstream process development and innovations in scalable equipment. All queries and end-user support Next, we investigated the priming effect of one treatment on the stimulatory response to the other. Taken together, our work highlights the significance of IFN--LPS synergy, leading to CCL2 expression in monocytic cells via a mechanism involving H3K27 acetylation. IFN- and TNF- are known to induce expression of several proinflammatory genes in macrophages. Group means between two data sets were compared using Students t-test and those of more than two data sets were compared using one-way ANOVA with post-hoc Tukeys test. The antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc.). For real-time PCR, each cDNA sample (50 ng/L) was amplified using Inventoried TaqMan Gene Expression Assay products including, GAPDH: Hs03929097_g1; CCL2: Hs00234140_m1, and STAT1: Hs01013996_m1 containing two gene-specific primers and one TaqMan MGB probe (6-FAM dye-labeled) using a TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) in Quant Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

11 , 1921 However, the underlying mechanism of CCL2 overproduction in obesity remains unclear. Tyrosine-phosphorylated STAT1 molecules dissociate from the receptor complex and undergo SH2 domain-mediated dimerization. The field of neuroscience continues to offer groundbreaking insight into the most complex biological systems. Total RNA was extracted and Ccl2 mRNA expression was quantified by real-time RT-PCR. On the contrary, given that histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induces the histone acetylation and increases gene expression, 39 we asked whether TSA could promote CCL2 expression by mimicking the effect of IFN- and substituting it in the synergistic response with LPS. Figure 3 H3K27 acetylation levels at different sites of CCL2 promotor region. 28 Regarding the intracellular events, IFN- binds to a hetero-oligomeric (IFNGR1/IFNGR2), class II cytokine receptor and activates a Jak-Stat signaling pathway implicating the Janus family kinases (Jak1 and Jak2) and the transcription factor called signal transducer and activator of transcription 1 (STAT1). Software Repository at the European Bioinformatics Institute (EBI), The FTP site for Software Repository at The European Bioinformatics Institute (EBI), The world's largest open-source software development Web site. The blots were probed with rabbit anti-human antibody against H3K27, Histone H3, p-STAT1 or STAT1 in 1:1000 dilution at 4C overnight.

Buy and sell today! Combined exposure to IFN-/LPS resulted in a further increase in CCL2 gene expression and protein release ( Figure 1A and B ). Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2CT method.

45 Another contributory factor in this synergy may be the strong stimulatory signaling co-induced by LPS and IFN- via the NF-B pathway. To test whether IFN- and LPS could synergistically induce the expression of CCL2, THP-1 monocytic cells were exposed to IFN- and LPS, alone or in combination, the treatment with the vehicle served as control.

(B) Western blot band densities were quantified and data were expressed as mean SEM (n = 3) values which were compared for various treatments using one-way ANOVA and post-hoc Tukeys test. The use of all the resources provided herein and any associated materials is at user's Next, we found that IFN--priming followed by LPS stimulation, but not LPS-priming followed by IFN- stimulation, was a more potent inducer of CCL2 in monocytic cells. # DY 279, R&D Systems Inc.). Western Blotting Materials and Methods Acetylation of H3K27 was analyzed by Western blotting. Elevated CCL2 gene (A) and secreted protein (B) expression is shown in THP-1 monocytic cells co-stimulated with IFN- and LPS compared to cells treated with IFN- and LPS alone. ELISA siRNA-Mediated Genetic Suppression of STAT1 Relative changes in Ccl2 gene expression were calculated using 2CT method and expressed as fold change over its expression in control (vehicle treatment). Shop new, used, and refurbished equipment on LabX and browse products for sale from hundreds of vendors. CCL2 protein was determined by ELISA.

(C) The schematic diagram of CCL2 gene promotor region is shown. third party. terms of use. Cells were incubated in RPMI medium containing 10%-FBS in a humidified incubator at 37C and 5% CO2. The quantity of isolated RNA was determined by using Epoch Spectrophotometer System (BioTek, Winooski, USA). our sites and services. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. CBI websites generally use certain cookies to enable better interactions with our sites and services. Relative CCL2 mRNA expression was expressed as fold change over average of control (vehicle treatment) gene expression.

In addition to monocytic cells and primary monocytes, THP-1-derived macrophages co-stimulated with IFN- and LPS also display elevated expression of CCL2 transcripts (D) and secreted protein (E). 62 Furthermore, we found that inhibition of acetyltransferases (HATs) by anacardic acid, which promote acetylation, resulted in suppression of the additive effect of IFN- and LPS on CCL2 production. Chromatin immunoprecipitation was done on cell lysates using H3K27ac antibody. However, inhibition of HDACs further enhanced the synergistic expression and production of CCL2 in response to IFN-/LPS.

tools. This research was funded by Kuwait Foundation for the Advancement of Sciences, grant number RA AM 2016-007. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as internal control to normalize the differences in individual samples. Wells for single or combined treatments were also incubated for 24 hrs simultaneously, with untreated well serving as control.

To evaluate whether the synergy between INF- and LPS, leading to amplified CCL2 expression in monocytes was dependent on STAT1, we transfected cells with STAT1 siRNA to silence STAT1 expression, which resulted in a more than 40% reduction of STAT1 mRNA levels compared to scrambled siRNA control (STAT1 mRNA: p = 0.0182) ( Figure 2A ). The lysates were clarified by centrifugation at 14,000 g for 10 min and supernatants were collected. They are not validated products and are provided "as is" and without warranty.

Tukeys test yielded pairwise comparisons among each of the groups using a conservative error estimate to find the groups that differed significantly from one another. Statistical Analysis Relative mRNA expression was expressed as fold expression over average of control gene expression. Alternatively, monocytic cells were primed by incubation with IFN- (10 ng/mL), washed and incubated with LPS (10 ng/mL) for 24 hrs.

Among chemokines, CCL2 and CXCL10 (IP-10) are known as IFN-regulated chemokines that function as potent attractors of monocytes, macrophages, T cells, natural killer (NK) cells, and antigen presenting dendritic cells (DCs) to the sites of inflammation, infection, or tissue damage. For all analyses, data from a minimum of three sample sets were used for statistical calculation. Applied Biosystems has filed 576 patents. The aim of this study was to investigate cooperativity between IFN- and LPS for augmenting CCL2 expression in monocytic cells/macrophages and to identify the underlying signaling mechanism involved. 51 In addition, it was also suggested that IFN- per se may have direct transcriptional effects. The data show that only the IFN- priming followed by LPS stimulation led to elevated CCL2 mRNA (F) and secreted protein (G) expression in monocytic cells compared to controls stimulated with IFN- or LPS alone. The data show significant suppression of (B) CCL2 mRNA (***p< 0.001) and (C) CCL2 secreted protein (**p< 0.01) in cells co-stimulated with IFN- and LPS as compared to those stimulated with IFN- or LPS alone.

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