Louis, MO), centrifuged at 2,000 rpm for 90 min at 30C, followed by incubation at 37C for an additional 6 h

Louis, MO), centrifuged at 2,000 rpm for 90 min at 30C, followed by incubation at 37C for an additional 6 h. formation by oxidized LDL. Taken together, these results illustrate a Jmjd3-dependent epigenetic regulatory mechanism for proinflammatory cytokine gene expression in SAA-stimulate macrophages. This mechanism may be subject to therapeutic intervention for sterile inflammation and atherosclerosis. 0111:B4 was purchased from Sigma-Aldrich (St Louis, MO). The inhibitors for protein kinases MEK (U0126) and PI3K (LY294002) were purchased from Calbiochem (San Diego, CA). The anti-Jmjd3 antibody was obtained from Abcam (Cambridge, MA). Antibodies for H3K27me3 and Jmjd3 (C-terminus) were obtained from Millipore (Billerica, MA). Antibodies for HDAC1, -actin, the anti-rabbit and anti-mouse IgG HRP linked antibodies were obtained from Cell Signaling Technology (Danvers, MA). 2.3 Cells preparation and culture Mouse macrophages (BMDMs and PMs) were prepared from WT or knockout C57BL/6 mice as described [45]. Human monocytic THP-1 cells (TIB-202), mouse RAW264.7 macrophages (TIB-71), the viral packaging cell line BOSC23 (CRL-11270) were all obtained from ATCC (Manassas, VA). The cells were maintained in Evacetrapib (LY2484595) RPMI 1640 supplemented with 2 mM of L-glutamine, 10% of FBS (GIBCO), 25 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell cultures were kept in a humidified atmosphere with 5% CO2 at 37C. 2.4 siRNA interference Mouse peritoneal macrophages were transfected Evacetrapib (LY2484595) with specific siRNA using Silencer? siRNA Transfection II Kit (Ambion) according to the manufacturer’s instructions. The cells were then recovered for 48 h before stimulation. The siRNA oligonucleotide were designed and synthesized by Shanghai RIBOBIO Co., LTD (Guangzhou, China). Sequence of MyD88-specific siRNA1 and Nonsense siRNA were shown in Supplementary table 1. 2.5 Plasmid constructs Mouse cDNA coding for Jmjd3, JmjC (a.a. 1141-1614) and JmjC carrying an Ala Casp3 mutation at His-1388 (Mut. JmjC) were a gift from Prof. Gioacchino Natoli (European Institute of Oncology, Milan, Italy) as described in [24]. The cDNAs were subcloned into the multi-cloning sites of a retrovirus-based expression plasmid, MigR1, which also contains an internal ribosome entry site (IRES) for GFP expression (Addgene, Cambridge, MA). Oligonucleotides targeting mouse Jmjd3 were annealed and ligated into the RNAi-Ready pSIREN-RetroQ ZsGreen vector (Clontech, Mountain View, CA). All sequences for Jmjd3 cloning and shRNA were shown in Supplementary table 1. 2.6 Retrovirus-mediated gene transfer BOSC23 cells were co-transfected with 6 g of the constructed plasmid plus 1.5 g of the pVSV-G plasmid (Clontech, Mountain View, CA) using HG TransGene Reagent (Health & Gene, China). After 6 h, the medium was removed and replaced with fresh medium. Viral supernatants were collected, passed through a filter and concentrated. For infection, cells were incubated with serially diluted retroviral supernatants in the presence of 8 g/ml Polybrene (Sigma, St. Louis, MO), centrifuged at 2,000 rpm for 90 min at 30C, followed by incubation at 37C for an additional 6 h. The media was replaced with fresh RPMI 1640 supplemented containing 10% FBS. After 48 h, the cells were treated with SAA for the indicated times, and then harvested for different assays. 2.7 Immunofluorescence RAW264.7 cells were grown on Microscope cover glass (Thermo-Fisher) and fixed with 4% paraformaldehyde at 4C. After washing and permeabilization, cells were inversed on the dilution of an anti-H3K27me3 antibody (10 g/ml) for overnight at 4C. The cells were then repeatedly washed with PBS and incubated with 20 g/ml of Alexa Evacetrapib (LY2484595) Fluor? 568 Goat Evacetrapib (LY2484595) Anti-Rabbit IgG (H+L) Antibody (Life technologies, Carlsbad, CA) for 60 min. Nuclei were stained with DAPI (10 g/ml) for 5 min. The cover glass was washed with PBS and examined under a Leica TCS SP UV confocal laser scanning microscope (Leica, Wetzlar, Germany). 2.8 Chromatin immunoprecipitation assay Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit (Millipore) according to the manufacturer’s with minor modifications. Briefly, after SAA stimulation, RAW264.7 cells were cross-linked and then washed and resuspended in SDS Lysis Buffer. Nuclei were fragmented by sonication. Chromatin fractions were cleared with protein A-agarose beads followed by immnoprecipitation overnight with an anti-H3K27me3 antibody (Millipore) or with control IgG. Cross-linking was reversed, followed by proteinase K digestion. The primer sequences were shown in Supplementary Table 1. Data are presented as the amount of DNA recovered relative to the input control. 2.9 Bone marrow transplantation To induce chimeras, male C57BL/6 mice (6 to 8 8 weeks of age) were exposed to a split exposure of 7 Gy total body irradiation, using an RS 2000 X-ray Irradiator (Rad Source Technologies, Suwanee, GA). One day before transplantation, bone marrow-derived cells were isolated from C57BL/6 mice, followed by infection with retrovirus expressing either scrambled shRNA or Jmjd3-specific shRNA (jmjd3sh2). After 24 h, bone marrow-derived cells were harvested and injected into.