site you are consenting to these choices. The data show significant suppression of STAT1 mRNA expression in cells transfected with STAT1 siRNA compared to control siRNA transfected cells (**p< 0.01). Shop new, used, and refurbished equipment on LabX and browse products for sale from hundreds of vendors. 34 , 54 The 3 most popular patent topics include: Molecular biology, Fluid dynamics, Biotechnology, DNA sequencing, Genetics, Nadeem Akhter,1 Shihab Kochumon,1 Amal Hasan,1 Ajit Wilson,1 Rasheeba Nizam,2 Ashraf Al Madhoun,2,3 Fatema Al-Rashed,1 Hossein Arefanian,1 Fawaz Alzaid,2,4 Sardar Sindhu,1,3 Fahd Al-Mulla,1 Rasheed Ahmad1 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as internal control to normalize the differences in individual samples. Now you can shop LabX to find antibodies, reagents, kits, and related tools for your research and clinical needs. Peripheral blood mononuclear cells (PBMCs) were isolated using HistoPaque density gradient method as described. 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. For real-time RT-PCR, cDNA (50 ng/L) was amplified using TaqMan Gene Expression Master Mix 4369016 (Applied Biosystems, CA, USA) and target gene-specific TaqMan Gene Expression Assays (Applied Biosystems, CA, USA) containing forward and reverse primers and a target-specific TaqMan minor groove binder (MGB) probe labeled with 6-fluorescein amidite (FAM) dye at 5 end and non-fluorescent quencher (NFQ)-MGB at 3 end of the probe, with 40 cycles of PCR amplification using 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA). Alternatively, monocytic cells were primed by incubation with IFN- (10 ng/mL), washed and incubated with LPS (10 ng/mL) for 24 hrs.

Methods: Monocytes (Human monocytic THP-1 cells) were stimulated with IFN- and LPS. dna analyzer applied biosystems labx Applied Biosystems is not responsible for providing any end-user support. 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. Who are the investors of Applied Biosystems? THP-1 monocytic cells were treated with anacardic acid (HATs inhibitor; 50 M) overnight or with TSA (HDACs inhibitor; 25 nM) for 6 h, followed by treatments with IFN- (10 ng/mL) and/or LPS (10 ng/mL) for 24h, and the treatment with vehicle alone served as control. 45 Another contributory factor in this synergy may be the strong stimulatory signaling co-induced by LPS and IFN- via the NF-B pathway. STAT1 Deficiency Diminishes the Synergy Between IFN- and LPS for CCL2 Expression 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. Since both IFN- and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity. CCL2-secreted protein levels were measured in the supernatants of THP-1 cells stimulated with IFN- (10 ng/mL), LPS (10 ng/mL), alone or in combination, using human CCL2 DuoSet ELISA kit and following the manufacturers instructions (Cat. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Acetylation of H3K27 was analyzed by Western blotting. Statistical Analysis Figure 1 IFN- and LPS cooperatively induce CCL2 in monocytic cells/macrophages. 63 It could be possibly a cell-type dependent mechanism that differentially regulates an epigenetic switch in monocytes vs effector T lymphocytes. The chromatin was reverse crosslinked and DNA from the ChIP fraction was purified using the spin column method. Relative STAT1 mRNA expression was expressed as fold expression over average of control (scrambled siRNA) gene expression. genetic seqstudio biosystems analyzer biosystems applied solid analyzer genetic flash sorry player Cell lysates were used for determination of H3K27 acetylation by Western blotting. Protein concentration was measured using QuickStart Bradford Dye (Bio-Rad Laboratories). 57 Like IFN-, LPS stimulation of macrophages also induces STAT1 phosphorylation which is required for promoting optimal expression of LPS-inducible genes. 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-. Relative mRNA expression was expressed as fold expression over average of control gene expression. 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. seqstudio sequencing fisher labcompare analyzer genetic sequencer biosystems sanger automated trade thermofisher amplification sequenzierung Inhibition or Activation of Acetylation by Pharmacological Agents Affects the Synergy Between IFN- and LPS 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. Get answers. What is Applied Biosystems's latest funding round? 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. It was shown that IFN- primes the regulatory elements via recruitment of STAT1 and the HATs that promote histone acetylation. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2CT method. (A) THP-1 monocytic cells were transfected with control/scrambled siRNA or STAT1 siRNA and incubated for 40h. biosystems applied labx 34 All p-values < 0.05 were considered significant (**p<0.01, ***p<0.001). All p-values < 0.05 were considered significant. As expected, we found a synergic upregulation of CCL2 protein expression in macrophages, following co-stimulation with IFN- and LPS ( Figure 1D and E ). Read our FAQs. Funding thermocycler cycler biosystems are intended for convenience only and do not imply endorsement or approval by Applied 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. CCL2 gene expression was determined by real-time RT-PCR. Overall, these data show that IFN- treatment induces the H3K27 acetylation, which enhances the accessibility of the CCL2 promoter. (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.

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed on the journal to which the article will be submitted; gave final approval for the version to be published; and agreed to be accountable for all aspects of the work. Applied Biosystems Participates in BioIT Alliance by Microsoft. This research was funded by Kuwait Foundation for the Advancement of Sciences, grant number RA AM 2016-007. our sites and services. (C) The schematic diagram of CCL2 gene promotor region is shown. limiting the foregoing, your use of such resources is subject to any applicable license or However, inhibition of HDACs further enhanced the synergistic expression and production of CCL2 in response to IFN-/LPS. A standard curve was generated by plotting the mean absorbance for each standard on the Y-axis against the concentration on the X-axis to draw a best fit curve through the points on the graph. Investors of Applied Biosystems include Life Technologies Corporation, PerkinElmer, Asset Management Ventures, Mayfield and U.S. Venture Partners. Combined exposure to IFN-/LPS resulted in a further increase in CCL2 gene expression and protein release ( Figure 1A and B ). Total RNA was purified from cells lysed in RLT buffer, using RNeasy kit and following the manufacturers instructions (Qiagen, Valencia, CA; USA). 35 PBMCs were seeded in 6-well plates (Costar, Corning Incorporated) at a cell density of 3 106 cells per well and cultured in a starvation (serum-free) medium at 37C for 3 h. Non-adherent cells were removed by gentle repeated washing with a serum-free culture medium and then adherent monocytic cells were removed and incubated for 24 h in RPMI medium containing 2% FBS. 60 We found that H3K27 acetylation was significantly higher at Ccl2+01A and Ccl2-03A transcription start sites in CCL2 promoter. Immunoreactive bands were developed using an Amer sham ECL Plus Western Blotting Detection System (GE Health Care, Buckinghamshire, UK) and visualized by ChemiDoc MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA).



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