Resistin

FLSs were seeded in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) in 10-cm dishes (1 × 10⁶ cells/dish), and were then incubated with 1000 ng/ml resistin (PeproTech) for 18 h. Total RNA was isolated from FLSs using TRIzol® (Invitrogen) according to the manufacturer’s instructions. RNA samples were digested with an RNase-free DNase set (Qiagen) to remove genomic DNA and further purified using the RNeasy kit (Qiagen). The quality of RNA samples was examined by the Agilent 2100 Bioanalyzer (Agilent Technologies) using RNA 6000 NanoChips. RNA samples with an RNA integrity number higher than 7 were used in further analyses, including a RNA-Seq and real-time polymerase chain reaction (PCR).

S. pyogenes (T1 strain), S. aureus and E. coli were grown in liquid broth at 37 °C over night. The cultures were diluted 1:50 (S. pyogenes) and 1:180 for E. coli and S. aureus. In order to determine the antibacterial effect of HBP (R&D systems, Minneapolis, MN) and resistin (PeproTech, Rocky Hill, NJ), the bacterial cultures were exposed to different concentrations of the peptides. Final concentrations of 5 μg/ml, 2 μg/ml, 1 μg/ml and 0.1 μg/ml of HBP and 2 μg/ml, 1 μg/ml, 0.1 μg/ml of resistin (Innovagen, Lund Sweden) were tested. An optical density (OD) curve was generated based on the turbidity measurement at 420–580 mm, using a Bioscreen C over a time of 24 h. Measurements were made every 15 min and data exported to a PC. Data were analysed with GraphPad Prism version 6.0 for Mac (GraphPad software).

All saliva samples from patients with OSCC and controls were analyzed in duplicate with quantitative ELISA. The ELISA kit for heparin cofactor 2 (Cat. number: LS‐F13221) was purchased from LifeSpan Biosciences (Seattle, WA, USA), for resistin (Cat. number: KHP0051) from Thermo Fisher Scientific (West Palm Beach, FL, USA), and for complement C5 (Cat. number: ab125963) from Abcam (Branford, CT, USA). The concentration of the studied proteins in saliva was measured by the sandwich ELISA method according to the instruction provided by the vendor of each kit. Absorbance was measured at 450 nm, and concentrations were calculated based on the recorded 7‐point calibration curves.
First, the variation coefficient of the parallel measurements was calculated and those data having more than 25 CV % value were excluded from statistical analysis.

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).