Myoblast cells isolated from EOM of normal subjects and patients (non-GO [n = 4] and GO [n = 7]) were grown to confluence in six-well plates in Media 199/FBS. Upon confluence, myoblasts were treated individually with or without IFN-γ (5000 U/mL), IGF-1 (320 ng/mL), IL-1β (20 ng/mL), and TNF-α (40 ng/mL) for 24 hours. For the drug treatment, myoblasts were treated with TGF-β (2 ng/mL) alone or TGF-β (2 ng/mL) plus either celecoxib (100 μM) or pioglitazone (28 μM) for 24 hours. Then, the cell culture supernatants were collected and concentrated using centrifugal filter system (Centricon-10; Amicon, Beverly, MA, USA). Cells were extracted with 0.5% SDS in the presence of protease inhibitor complex containing antipain-hydrochloric acid, bestatin, chymostatin, E-64, leupeptin, pepstatin, phosphoramidon, pefabloc SC, ethylenediaminetetracetic acid, and aprotinin (Boehringer Mannheim Biochemicals; Penzberg, Upper Bavaria, Germany). The cell supernatant and cell lysate samples were mixed with ×2 SDS gel-loading buffer (100 mM Tris-Cl [pH 6.8], 4% [wt/vol] SDS, 0.2% [wt/vol] bromophenol blue, 20% [vol/vol] glycerol, 200 mM dithiothreitol) and heated at 95°C for 4 minutes. An equivalent amount of total proteins at 20 μg/lane was loaded onto gels (4% acrylamide stacking gel and 12% acrylamide resolving gel). Gel electrophoresis was performed with a standard buffer system at a constant voltage of 90 V for 2 hours. Gels were presoaked in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) before electrophoretic transfer (at 90 mA for 3 hours) of proteins onto nitrocelluloses. Nitrocellulose membranes were incubated in blocking buffer containing 1X Tris-buffered saline (TBS; 10 mM Tris, pH 7.5, 150 mM sodium chloride), 0.05% Tween 20, and 5% nonfat dry milk for 3 hours at 37°C. After that, primary antibodies (COX-2, PPAR-γ, HAS1, HAS2, and HAS3 diluted at 1.0 μg/mL in 1X TBS and 1% nonfat dry milk) were incubated with the membranes overnight at 4°C. After being washed, nitrocellulose membranes were incubated with respective alkaline phosphatase–conjugated immunoglobulin (1 μg/mL) for 2 hours at room temperature. After washes with TBS/0.05% Tween 20, proteins were visualized using nitroblue tetrazolium and bromochloroiodindolyl phosphate chromogenic substrates in developing buffer (100 mM Tris, pH 9.5, 100 mM sodium chloride, 500 mM magnesium chloride). Protein sizes were determined by comparison with a protein molecular weight marker (Amersham Life Science, Braunschweig, Germany). For quantitation, immunoblots of COX-2, TSH-R, HAS1, HAS2, and HAS3 were measured by scanning densitometry using a dual-wave length densitometer (Shimadzu TCL Scanner; Shimadzu Scientific, Columbia, MD, USA) connected to a data recorder (Shimadzu DR2; Shimadzu Scientific). Band intensities were quantitated and were normalized to the corresponding levels of Actin using commercial software (Phoretix 1D Advanced, version 4.01; Phoretix International, Newcastle upon Tyne, UK).