Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Penicillin Streptomycin Solution, 2 mM L-glutamine, Lipofectamine 2000 and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture dishes, 6-well and 12-well plates were purchased from Greiner Bio-One (Frickenhausen, Germany). Polyvinyldifluoride (PVDF) membrane and an Immobilon Western Chemiluminescent HRP Substrate detection system were purchased from Millipore (Billerica, MA, USA). All of primary antibodies specific for IL-6 (sc-28343), CXCR2 (sc-32780), c-Raf (sc-7267), MEK (sc-6250), ERK (sc-514302), JNK (sc-7345), p38 (sc-81621), c-Jun (sc-166540) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal rabbit antibodies specific for phosphorylated forms of c-Raf (#9427), MEK (#3958), ERK (#4376), JNK (#9255), p38 (#9216) and c-Jun (#2361) were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). All inhibitors against signal pathway components were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human CXCL1 was obtained from PeproTech (Rocky Hill, NJ, USA). All small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Written informed consent was obtained from all study participants, and the study was approved by the Institutional Review Board of Shin Kong Wu Ho-Su Memorial Hospital (20140712R). Primary SFs were isolated from tissue with OA patients who received knee replacement surgery, using previously described methods (17, 18). The NSFs and RASFs were purchased from Cell Applications, Inc. (San Diego, CA, USA) and Riken cell bank (Ibaraki, Japan), respectively. Fresh synovial tissues were finely minced and digested in DMEM containing 2 mg/ml type II collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C and under 5% CO2. The SFs were maintained in complete DMEM with 10% FBS, Penicillin Streptomycin Solution, and 2 mM L-glutamine at 37°C with 5% CO2.
Real-time quantitative polymerase chain reaction (qPCR)
Cells grown in 6-well plates were treated as described in the Figure legends, and total RNA was extracted from the cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The quantity and purity of RNA were assessed using a Nanodrop ND 1000 (Thermo Fisher Scientific, Wilmington, DE, USA). The quality of RNA was further examined by agarose gel electrophoresis. Subsequently, 1 μg mRNA were subjected to reverse transcription to produce complementary DNA (cDNA).
qPCR was prepared using SYBR Green (KAPA Biosystems, Woburn, MA, USA), according to the manufacturer’s protocol. The primers used in qPCR analysis (human IL-6, CXCL1, and GAPDH) were purchased from Sigma-Aldrich. Reactions were carried out in triplicate using StepOnePlus (Applied Biosystems, Foster City, CA, USA). The cycling conditions were 10 min of polymerase activation at 95°C followed by 40 cycles at 95°C for 15 s and at 60°C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification, to calculate the cycle number at which the transcript was detected (denoted as CT).
Western blot analysis
The total cell lysates were extracted from the cells grown in 6-well plates and treated as described in the Figure legends. Proteins were resolved using SDS-polyacrylamide gel electrophoresis and transferred to Immobilon PVDF membranes. The blots were blocked with 5% BSA for 1 h at room temperature and then probed using primary antibodies (1:5000 for β-actin; 1:1000 for all of the others) for 1 h at room temperature. After three washes, the blots were incubated with secondary antibodies (1:1000) for 1 h at room temperature. Finally, the blots were photographed with enhanced chemiluminescence using a ChemiDoc-It® Imaging System (UVP Inc., Upland, CA, USA). Quantitative data were obtained using ImageJ software (National Institutes of Health, USA). Each band was separately selected and with the ROI tool and “Gels” function, followed by quantification of peak area of obtained histograms. The densitometric data of IL-6 protein was normalized to b-actin and the other phosphorylated proteins were normalized to corresponding total proteins, respectively.
Cells grown in chamber slides were subjected to immunofluorescence staining. In brief, the treated cells were fixed with 4% paraformaldehyde at room temperature. Thirty minutes later, 5% nonfat milk in phosphate buffer saline (PBS) containing 0.25% Triton X-100 was added to the cells. The cells were then incubated in rabbit anti-c-Jun (1 : 100) and fluorescein isothiocyanate- (FITC)-conjugated goat anti-rabbit secondary antibodies (1:500; Leinco Technology Inc., St. Louis, MO, USA) for 1 h. FITC was detected using a Zeiss fluorescence microscope.
All values are reported as the mean ± standard error of the mean (SEM). A statistical comparison between two samples was performed using the Student’s t-test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference (LSD) post hoc test. In all comparisons, p < 0.05 was considered significant.