After the proteins were eluted with elution buffer, 1?M Tris buffer was added to neutralize the additional acid. and, in turn, activate the Akt/NF-B signaling pathway. The nuclear localization of p65 promoted the expression of NLRP3 and pro-IL-1, resulting in priming. Moreover, ACPA stimulation activated pannexin channels, leading to ATP release. The accumulated ATP bound to the P2X7 receptor, leading to NLRP3 inflammasome activation. Conclusions Our study suggests a new hypothesis regarding IL-1 production in RA involving ACPAs, which may be a potential therapeutic target in RA treatment. scores) were calculated to indicate the expression of the target molecules. Briefly, the score was calculated by multiplying the percentage of positive cells per slide (0C100%) by the dominant staining intensity pattern (1, negative or trace; 2, weak; 3, moderate; and 4, intense). Samples with scores of 0C200, 201C300, and 301C400 were classified as having negative or low, intermediate, and high expression, respectively.43 Cell preparation Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum under standard culture conditions (37?C and 5% CO2). PBMCs were isolated Cefmenoxime hydrochloride by density gradient centrifugation over Ficoll-Hypaque (Lymph prep, Oslo, Norway) from informed, healthy volunteers. Primary monocytes were isolated from human PBMCs using an EasySep? Human Monocyte Enrichment Kit without CD16 Depletion (Stemcell Technologies, Ontario, Canada) and were differentiated by granulocyte macrophages colony-stimulating factor treatment (Pepro Tech, NJ, USA) for 6 days. THP-1 cells were purchased from the American Type Culture Collection (ATCC, VA, USA) and were differentiated overnight with 50?ng/ml PMA. After differentiation, cells were stimulated with 100?g/ml ACPA for 12?h. For the stimulation and inhibition studies, cells were pretreated in a 37?C incubator with the following compounds for the indicated time periods and at the indicated concentrations: ACPA IgG (0-200?g/ml, 0-12?h), control IgG (100?g/ml, 12?h), LPS (100?ng/ml, 3?h), ATP (5?mM, 1?h), GRGDS (50?M, 30?min), TAK242 (100?nM, 2?h), Bay-117082 (10?M, 30?min), glibenclamide (50?M, 30?min), probenecid (250?M, 12?h with stimulation), and oxATP (300?M, 30?min). Short hairpinRNA- and small interfering RNA (siRNA)-mediated knockdown To knock down CD147 expression, we used the previously constructed trans-lentiviral pLKO system.44 The plasmids in the trans-lentiviral pLKO system, including psPAX2 (1.125?g/ml), pMD2G (0.125?g/ml), and pLKO A6/NC (1.25?g/ml), were transfected into HEK 293T cells using Lipofectamine TNRC23 2000 (Invitrogen, Basel, Switzerland) according to the manufacturers instructions. The culture medium containing lentivirus was collected and added to PBMC-derived monocytes and THP-1 cells for 48?h. Puromycin Cefmenoxime hydrochloride at 2?g/ml (Sigma, Buchs, Switzerland) was added to the medium for further selection. To knock down NLRP3 and integrin 1 Cefmenoxime hydrochloride (ITGB1) expression, we purchased siRNA from GenePharma (Shanghai, China). After differentiation in 6-well plates, PBMC-derived monocytes and THP-1 monocytes were transfected with control siRNA (100?nM), NLRP3 siRNA (100?nM), or integrin 1 siRNA (100?nM) for 96?h using Lipofectamine 2000. Immunoblotting and immunoprecipitation After different treatments, cell culture medium was collected and centrifuged at 4?C for 5?min at 2000??and 4?C for 20?min, the supernatant was collected, and protein was quantified with a bicinchoninic acid protein assay (Thermo Scientific, Rockford, USA). Equal amounts of protein were removed and boiled for 8?min in Laemmli buffer before being resolved on 10% or 12.5% polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked in 5% skim milk or 3% bovine serum albumin (BSA; for phosphoproteins) and incubated with the corresponding primary antibodies overnight at 4?C. After three washes (5?min each) with TBST, membranes were incubated with an HRP-conjugated secondary antibody for 1?h at room temperature (RT). Signals were detected with a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) using Pierce? ECL Western Blotting Substrate (Thermo Fischer Scientific, CA, USA). Each experiment was performed at least three times. Coimmunoprecipitation was performed using a PierceTM coimmunoprecipitation kit (Thermo Scientific, MA, USA), which utilizes capture antibodies covalently bound directly to beads. After the proteins were eluted with elution buffer, 1?M Tris buffer was added to neutralize the additional acid. Protein quantification was carried out before the immunoblot assay. Anti-CCP and IL-1 detection Patient blood samples were collected and stored Cefmenoxime hydrochloride at 4?C before detection. Anti-CCP titers were evaluated with an anti-CCP assay (Euroimmun, Lbeck, Germany). Before measuring IL-1 production in cells, the culture medium was replaced with serum-free medium. After stimulation, the medium was collected and centrifuged at 10,000??for 5?min. IL-1 production was measured with.