Mpges 1

MTT and L-glutamine were obtained from USB (Cleveland, OH, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (São Paulo, SP, Brazil). Gentamicin was purchased from Schering-Plough (Whitehouse Station, NJ, USA) and DMSO was purchased from Amresco (Solon, OH, USA). Mouse mAb anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and polyclonal antibodies against COX-1, COX-2 and mPGES-1 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). PGE₂; PGI₂ enzyme immunoassay kit; and the compounds SC-560 NS-398, pyrrolidine-2 (PYR-2), FKGK11, KH064, and Batimastat (BB-94) were also purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Secondary antimouse and antirabbit antibodies conjugated to HRP and nitrocellulose membrane were obtained from GE Healthcare (Buckinghamshire, UK), while the leptin, resistin, and adiponectin immunoassay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The cytometric bead assay (CBA) kit was purchased from BD Bioscience (San Jose, CA, USA).

Protein was extracted from cells with a buffer containing 1% SDS, 100 mM Tris-HCl, 1 mM PMSF, and 0.1 mM β-mercaptoethanol, following treatment with 0–2.5 μg/ml rat recombinant MIF (ProSpec) for 24 h. Alternatively, protein was extracted from 1 cm spinal segments of injured site at 0 day, 1 day, 4 days, and 1 week following contusion (n = 8 in each time point). Protein concentration of each specimen was detected by the Bradford method to maintain the same loads. Protein extracts were heat denatured at 95 °C for 5 min, electrophoretically separated on 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were subjected to the reaction with a 1:1000 dilution of primary antibodies in TBS buffer at 4 °C overnight, followed by a reaction with secondary antibody conjugated with goat anti-rabbit or goat anti-mouse HRP dilution 1:1000 (Santa Cruz) at room temperature for 2 h. After the membrane was washed, the HRP activity was detected using an ECL kit. The image was scanned with a GS800 Densitometer Scanner (Bio-Rad), and the data were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-actin (1:5000) was used as an internal control. Antibodies used in Western blot are MIF, COX1, cPGES (Abcam); COX2, mPGES-1, mPGES-2 (Cayman); CD74 (Biorbyt); and β-actin (Proteintech).

Cell lysates were prepared using RIPA lysis buffer (Millipore, MA, USA) with protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IL, USA). Protein content was measured using a bicinchoninic acid assay kit (Thermo Scientific). Total protein (40 μg) was separated using 12.5% SDS-PAGE gel electrophoresis and electrotransferred to a polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with primary antibodies at the following concentrations: COX-2 (1:1000, BD Biosciences, Heidelberg, Germany), mPGES-1 (1:500, Cayman Chemicals), and β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies (1:5000) goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology) were used for blotting. Chemiluminescent signals were generated via a chemiluminescence reagent (Thermo Scientific) and captured using a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Surgically resected human colon tissues at all clinical stages were fixed in 10% formalin overnight at room temperature. For histology, fixed tissues were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) according to standard protocols. Immunohistochemistry staining for human cyclooxygenase-2 (COX-2, #12282, Cell Signaling Technology; dilution 1:200), mPGES1 (#160140, Cayman Chemical Company; dilution 1:50), TBXAS1 (#160715, Cayman Chemical Company; dilution 1:50), TBXA2R (#10004452, Cayman Chemical Company; dilution 1:50), or Ki-67 (RM-9106, Thermo Scientific, Fremont, CA; dilution 1:200) was performed using an ABC complex kit (PK-6100, Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. Sections were counterstained with Harris's hematoxylin. For antibody-negative controls, the primary antibodies were substituted with normal rabbit serum. Immunohistochemistry staining intensity was quantified by calculating the integrated optical density (IOD, sum) of the area of interest using the Image Pro-Plus 7.0 software program.

The total protein in the kidney tissue was extracted by a lysis buffer and the concentrations were determined through the Coomassie reagent. Equal amounts of the tissue protein (50 μg) were denatured at 100°C for 10 min, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for 1 h with rabbit polyclonal antibody, raised against mouse COX-2 and mPGES-1 (Cayman Chemical, MI, USA). The blots were washed with TBS followed by incubation with a horseradish peroxidase-conjugated secondary antibody and the immune complexes were detected using the ECL system (Amersham). The Bio-Rad electrophoresis image analyzer (Bio-Rad, UK) was used to quantify the protein signals.

UA was bought from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin solution (EDTA) were bought from Gibco (Invitrogen, Grand island, NY). COX-2 (catalog no. 160106) and mPGES-1 (catalog no. 160140) antibodies were bought from Cayman Chemicals (Ann Arbor, MI). Cyclin A2 antibody (catalog no. ab7956) was purchased from Abcam (Cambridge, MA). Cyclin D1 antibody (catalog no. 2978) and GAPDH antibody (catalog no. ab9485) were purchased from Cell Signaling Technology (Danvers, MA). The PGE₂ enzyme immunoassay kit (catalog no. 514010-96) was provided by Cayman Chemicals (Ann Arbor, MI). COX-2 inhibitor NS-398 (catalog no. s1772) was from Beyotime (Shanghai, China).