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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. Human primary monocytes were isolated from peripheral blood samples collected from healthy individuals after their written informed consent, and the study was approved (Protocol #: RA-2010-003) by ethics committee of Dasman Diabetes Institute, Kuwait. 
Figure 1 IFN- and LPS cooperatively induce CCL2 in monocytic cells/macrophages. 
was read using a microplate reader at 450 nm, with wavelength correction set to 540 nm or 570 nm. From pipettors and pipette tips, to CO2 incubators, fume hoods and cell imaging systems, you can shop all the essentials of the modern cell culture laboratory on LabX. 29 , 30 All p-values < 0.05 were considered significant (**p<0.01, ***p<0.001). In contrast to this inflammatory gene (CCL2) induction by H3K27 acetylation, H3K27 trimethylation (H3K27m3) induced repression of the anti-inflammatory pathway genes (MERTK, PPARG, and RANK).
Biosystems and we neither support nor make any warranty for such resources. 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. 
STAT1 molecules are associated with the paired docking sites, being in close proximity to activated Jaks1/2 and are activated by phosphorylation on a single tyrosine residue (Y701).
By continuing to use this site you are consenting to these choices. All p-values < 0.05 were considered significant (*p< 0.05 and **p< 0.01).
11 , 16 , 17 Functionally, CCL2 interacts with chemokine receptor 2 (CCR2) on the surface of monocytes and initiates their tissue infiltration. The major STAT protein activated by IFN- is STAT1, and many IFN- functions are mediated through direct activation of immune effector processes by STAT1. Although there exists some conflicting data on levels of proinflammatory cytokines expressed in human obesity, 22 , 23 the levels of both LPS and IFN- have often been found to be increased in obese individuals, 2427 but whether or not they inter-play to regulate the expression of CCL2 in monocytic cells remains to be investigated. Proinflammatory M1-like polarization of macrophages requires a pro-oxidant Th1 microenvironment, marked by two initiation signals, such as IFN- and LPS or TNF-. All p-values < 0.05 were considered significant (ns, non-significant, *p< 0.05, **p< 0.01, and ***p< 0.001).
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. Browse ads and contact the seller directly or request a quote for more details.
11 , 1921 However, the underlying mechanism of CCL2 overproduction in obesity remains unclear.
The antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc.).
ELISA All data are expressed as mean SEM (n 3) and group means between two data sets were compared using Students t-test. All p-values <0.05 were considered significant (ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). 
Our results further provide a critical mechanism of IFN--dependent epigenetic regulation, in which H3K27 acetylation instructs the synergic crosstalk between IFN- and LPS to drive expression of CCL2 in monocytic cells, suggesting a new target for treating metabolic inflammation. Comments regarding this site and Applied Biosystems resources provided herein Obesity has spread by epidemic proportions, affecting a large number of people globally and placing them at high risk of developing several chronic metabolic diseases including type 2 diabetes (T2D), metabolic syndrome, cardiovascular disease and fatty liver. biosystems stepone applied labx Applied Biosystems BioTek Synergy repair ABI 7500, Applied Biosystems ABI StepOne, StepOne plus qPCR, Applied Biosystems ABI 7500 PCR Temperature Verifi, Applied Biosystems ABI ProFlex simpliamp Quantstud, Applied biosystems ABI 7500 Precision Plate Hold, ABI 4397094 Applied Biosystems 7500 Fast qPCR heat, Applied biosystems ABI 7500 / ABI 7500 FAST Real-t, ABI Applied BioSystems Life 7500 Fast Real-Time PC, ABI Applied BioSystems Life 7500 Real-Time PCR Blo, ABI 7500 pcr Applied Biosystems 7500 qPCR heated c, Applied Biosystems ABI StepOne Plus Heating cover, Applied Biosystems ABI Veriti 96 well 0.2ml block, Applied biosystems ABI StepOne Plus Real-time PCR, Applied Biosystems ABI 7500 or 7500 Fast Real-time, Applied Biosystems ABI pcr QuantStudio Dx Fast 96, Applied Biosystems Thermo ABI SeqStudio Genetic An, Applied Biosystems ABI Calibration kit 96well 0.1m, Applied Biosystems ABI 7500 or 7500 fast PCR power, Applied biosystems ABI 7500 8 Well strips Precisio, Applied biosystems ABI StepOne & StepOne Plus only, NewThermo Applied Biosystems StepOne & StepOne Plu, Applied biosystems Abi pcr veriti Left ear or Rig, New Applied Biosystems 384-Well Block for QuantStu, Unmatched speed and performance -- reproducible, specific and sensitive PCR results in just 25 minutes, Optimized for convenience -- integrated 96-well system designed for consistent fast results, Results you can count on -- extension of the family of proven PCR solutions. For transient transfection, THP-1 cells (1 106) were resuspended in Nucleofector solution and transfected separately using 30 nM STAT1 siRNA and scrambled negative control siRNA. Applied Biosystems by Life Technologies, The SOLiD System: Next-Generation Sequencing, Applied Biosystems Participates in BioIT Alliance by Microsoft.
Activated STAT1 translocates to the nucleus and binds to its cognate promoter DNA at the gamma interferon-activated site (GAS). IFN- is known to prime cells and potentiate the effects of LPS stimulation. 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. THP-1 cells were differentiated into macrophages as previously described. Author Contributions These findings clearly highlight the importance of acetylation in this cooperativity. C-C chemokine ligand (CCL)-2, also called macrophage chemoattractant protein (MCP)-1, plays a key role in recruiting monocytes, the precursors to macrophages that accumulate in adipose tissue during obesity. Synergistic amplification of CCL2 expression by co-stimulation was a plausible outcome of this study. was subtracted from each value. Alternatively, monocytic cells were primed by incubation with IFN- (10 ng/mL), washed and incubated with LPS (10 ng/mL) for 24 hrs.
Notably, CCL2 gene and protein expression and was significantly diminished in cells treated with anacardic acid before IFN-/LPS co-stimulation, compared to likewise stimulated controls without pre-treatment with anacardic acid ( Figure 4A and B ). biosystems termociclador cycler thermal cycler applied 2720 biosystems pcr cycle fisher thermo Total RNA was purified from cells lysed in RLT buffer, using RNeasy kit and following the manufacturers instructions (Qiagen, Valencia, CA; USA). In particular, histone acetylation has been shown to play important roles in upregulating CCL2 expression in inflammatory cells. including the software tools, is conditioned on your acceptance of the above terms,
1115 Keywords: CCL2, monocytes/macrophages, LPS, IFN-, H3K27 ac, inflammation The LabX marketplace has all the laboratory products, equipment, and accessories for your cell culture needs. Herein, we show that IFN- and LPS, the two inflammatory initiators of inflammation, may interact synergistically to enhance CCL2 expression in monocytes/macrophages.
Ccl2 mRNA and protein expression was quantified and statistically analyzed as described above. The optical density (O.D.) 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.
51 In addition, it was also suggested that IFN- per se may have direct transcriptional effects.
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All data were expressed as mean SEM values (n = 3) and group means between two data sets were compared using Students t-test. biosystems applied adhesive microamp clear film 
biosystems applied solid analyzer genetic flash sorry player The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR. array endogenous taqman biosystems applied human control our sites and services. IFN--Mediated H3K27 Acetylation is Involved in Synergistic Expression of CCL2 Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN- in this synergy. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Funding 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. Investors of Applied Biosystems include Life Technologies Corporation, PerkinElmer, Asset Management Ventures, Mayfield and U.S. Venture Partners.
(F and G) THP-1 cells were primed with IFN- and LPS, separately, followed by LPS and IFN- treatment, respectively, for 24h.
To measure CCL2 concentrations, averages of duplicate readings for each standard, control, and sample were calculated, and the average zero standard O.D.
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.
LabX keeps pace with this industry by offering a wide range of instruments and solutions for the most demanding questions. 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. Applied Biosystems is not responsible for providing any end-user support.
40 Our data show that stimulation of monocytes/macrophages with IFN- and/or LPS induces CCL2 expression.
Applied Biosystems's latest funding round is Merger. Your use of any resources provided herein, Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2CT method.
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 &. Veuillez vous connecter pour afficher les prix de votre compte et la disponibilit de vos produits. pcr applied biosystems plus step system stepone cycler machines thermal realtime (C) The schematic diagram of CCL2 gene promotor region is shown. We incubated monocytic cells with vehicle, IFN-, LPS, or IFN-+LPS for 4 hrs. 
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site you are consenting to these choices. After washing thrice, substrate solution was added (100 L/well) and plates were again incubated at room temperature for 20 min in the dark. Chemokines are small chemoattractant proteins that impact the trafficking of immune cells to the sites of inflammation. pcr biosystems applied system fast qpcr thermo fisher scientific biocompare quantitative instruments quote 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). Relative changes in Ccl2 gene expression were calculated using 2CT method and expressed as fold change over its expression in control (vehicle treatment). This is because macrophages are known transducers of IFNG signalling and are also first-responders to LPS via its target receptors TLR4/CD14. Chromatin Immunoprecipitation (ChIP) Assay 
