Statistical differences (a 0.05 or bfor 10 PF-05089771 min and supernatant assessed for matrix-metalloproteinase-9 (MMP-9) and tumor necrosis factor (TNF). cells were grown, cultured, and prepared for experiments in the University of Missouri-St. Louis Cell Culture Facility as per accepted guidelines. Cells were treated with LPS for selected exposure times and the conditioned medium was PF-05089771 collected for analysis of MMP-9 and TNF production. Inhibitors including DMSO, cAMP regulators, and anti-TNF antibody were added to the cells prior to LPS treatment. MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software. TNF secretion was analyzed by enzyme-linked immuno sorbent assay. All data is presented as the average and standard error for at least 3 trials. Statistical analysis was done using a two-tailed paired Student bacterial 026.B6 LPS (Sigma-Aldrich, St. Louis). Cells were incubated for 72 h at 37 C in 5% CO2. DMSO concentrations were varied from 0.016%-2% to evaluate the effect on cell response and specific concentrations for each experiment is detailed in each figure legend. Control cell treatments without LPS but in the presence of DMSO were done for all studies. For experiments assessing the regulatory role of cAMP on LPS-stimulated MMP-9 and PF-05089771 TNF secretion, THP-1 monocytes were treated with cAMP regulators dibutyryl 3′,5′-cyclic adenosine monophosphate (Bt2-cAMP), forskolin (Fsk), and 3-isobutyl-1-methylxanthine (IBMX) for 5 min prior to subsequent treatment with 1 g/mL LPS. Cells were incubated for 6-72 h at 37 C in 5% CO2. In one experiment, cells were stimulated with 0.3 g/mL LPS. Concentrations or concentration ranges used for the regulators were 0.3 mmol/L for Bt2-cAMP, 0.01-0.1 mmol/L for Fsk, and 0.01-0.3 mmol/L for IBMX. Control cell treatments without LPS but in the presence of cAMP regulators were done for all studies. For experiments assessing the effect of an anti-human TNF monoclonal antibody (MAB610, R&D Systems, Minneapolis, MN, United States) on LPS-stimulated MMP-9 and TNF secretion, THP-1 monocytes were treated with the antibody for 5 min prior to subsequent treatment with 1 g/mL LPS. Cells were incubated for 48 h at 37 C in 5% CO2. Control cell treatments with or without LPS and/or in the presence or absence of the antibody, were done for these studies. After all treatments, cells were removed from each well, centrifuged at 2500 g for 10 min, and the supernatant was collected and frozen at ?20 C for subsequent analysis. Western blot Supernatants from individual wells of treated THP-1 monocytes were mixed 1:1 with Laemmli SDS sample buffer (Bio-Rad) containing 5% -mercaptoethanol. Samples and 10 L recombinant human MMP-9 western blotting standard (WBC018, R&D Systems, Minneapolis, MN, United States) were heated at 95 C for 5 min and separated on 7.5% Tris-HCl gels (Ready Gel, Bio-Rad) under denaturing conditions (25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS at pH 8.3) using a Mini Protean 3 Cell (Bio-Rad). Pre-stained SDS-PAGE standards (BioRad) were loaded directly into gel wells. Gels were transferred to Immobilon-P PVDF membrane (Millipore) in transfer buffer containing 25 mmol/L Tris base, 192 mmol/L glycine, and 10% methanol at pH 8.3 using a Tank VEP-2 electroblotting system (Owl Separation Systems). Following protein transfer, the membrane was blocked for 1 h at 25 C with PBS containing 0.2% Tween 20 (PBST) supplemented with 5% nonfat dry milk, and probed for 1 h at 25 C with 1 g/mL goat anti-human MMP-9 antibody (AB911, R&D Systems) in PBST containing 1% milk. After washing membrane 3 in PBST, the membrane was incubated with a 1:1000 dilution of goat anti-mouse IgG-HRP secondary antibody (R&D systems) in PBST BSG containing 1% milk for 1 h at 25 C. Protein detection was accomplished using ECL Western Blotting Substrate reagents (Pierce).
