ns, not significant; *, 0.05; and ***, 0.0005 by Students = 5. of dTBP2 on mast cell degranulation and allergen-induced anaphylactic reactions. We found that bacterial product lipopolysaccharide increased the expression of dTCTP in mast cells and rapidly released dTCTP by the mast cell stimulator compound 48/80. Interestingly, the released dTCTP further promoted mast cell degranulation in an autocrine activation manner and increased calcium mobilization in mast cells, which is essential for degranulation. Furthermore, dTBP2 directly and dose-dependently inhibited mast cell degranulation enhanced by compound 48/80, suggesting a direct and potent anti-anaphylactic activity of dTBP2. dTBP2 Pyridone 6 (JAK Inhibitor I) also significantly suppressed the dTCTP-induced degranulation and histamine release through inhibition of the p38 MAPK signaling pathway and suppression Pyridone 6 (JAK Inhibitor I) of lysosomal expansion and calcium mobilization in mast cells. More importantly, administration of dTBP2 decreased mortality and significantly attenuated histamine release and inflammatory cytokine production in compound 48/80-induced systemic anaphylactic reactions. These results suggest that dTBP2 is beneficial for the control of anaphylaxis with increased dTCTP. anaphylactic Pyridone 6 (JAK Inhibitor I) reactions and mast cell degranulation, and further analyzed the molecular mechanisms underlying the inhibition of dTBP2 in mast cell degranulation. Materials and Methods Reagents Compound 48/80 (C48/80) was purchased from Sigma-Aldrich (St. Louis, MO, United States). The Dulbeccos modified Eagles medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Recombinant TCTP proteins r-dTCTP and r-mTCTP were isolated from the BL21 (DE3) PLysS strain that was transformed with PRSET A vector of His-tagged dTCTP and mTCTP. r-dTCTP and r-mTCTP were used for cell treatment after purification using Ni-NTA agarose (QIAGEN, Hilden, Germany) and anion Exchange Chromatography with HiTrap Pyridone 6 (JAK Inhibitor I) Q Column (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, United States), followed by endotoxin removal. dTBP2 Rabbit Polyclonal to GNB5 peptide was synthesized at the peptide synthesizer facility PepTron Inc. (Daejeon, Korea) and the purity of the synthetic peptide was confirmed to be 98%. Isolation and Cultivation of Bone-Marrow Derived Mast Cells) Bone marrow cell suspensions were isolated by flushing femurs and tibias from C57BL/6J mice (male, 8?weeks old), and cultured in RPMI-1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 50?M 2-mercaptoethanol, and recombinant murine IL-3 (rmIL-3, 10?ng/ml, BioLegend, San Diego, CA, United States) for 4?weeks (Liao et al., 2020). The purity of BMMCs assessed by staining and flow cytometry analysis with anti-FcRI (Invitrogen, Carlsbad, CA, United States) and anti-c-kit antibody (eBioscience, San Diego, CA, United States) confirmed that they are 95% pure. The BMMCs were Pyridone 6 (JAK Inhibitor I) cultivated between days 29 and 49 post-isolation and were used for all functional assays. -Hexosaminidase Assay Untreated BMMCs or those pre-treated with vehicle, r-dTCTP, and r-mTCTP for 24?h were centrifuges and the cell pellets were resuspended in extracellular buffer (10?mM HEPES, 137?mM NaCl, 2.7 mM KCl, 0.4?mM Na2HPO4.7H2O, 1.4?mM CaCl2, 1?mM MgCl2, 5.6?mM glucose, and 0.04% BSA), and treated with 50?g/ml C48/80 for either 5?min or indicated time. Untreated sample were prepared to use as controls. The cell supernatant was harvested and subjected to -hexosaminidase release assay using 4-nitrophenyl 2-acetamido-2-deoxy–D-glucopyranoside in 0.1?M citrate buffer (pH 4.2) (PNAG, 3.4?mg/ml, TCI Chemicals, Japan). Reaction was stopped with 0.4?M glycine buffer (pH 10.8), and optical density was measured at 405?nm. Immunofluorescence and LysoTracker Staining BMMCs were incubated with LPS (1?g/ml) for 24?h and treated with C48/80 (50?g/ml) for 5?min before harvest. Cells were fixed with 4% paraformaldehyde for 10?min at RT, permeabilized in 0.1% Triton X-100/PBS for 10?min, and incubated with antibody against lysosomal membrane-associated protein 1 (LAMP1, Santa Cruz Biotech Inc., Santa Cruz, CA, United States) and TCTP (Abcam, Cambridge, MA, United States), followed by staining with Alexa Fluor 488- or Alexa Fluor 555-coupled secondary antibodies (Invitrogen). Nuclei were stained with DAPI (1?g/ml). Cells were observed under a confocal laser scanning microscope (ZEISS LSM 880 with Airyscan, Oberkochen, Germany). For LysoTracker staining, cells were pre-treated with dTCTP (10?g/ml) and/or dTBP2 (100?g/ml) and incubated with LysoTracker? (75?nM, Thermo Fischer Scientific) for 1?h at 37C in.
ns, not significant; *, 0