broblasts to proliferate. After fibroblasts had grown out from the explants, monolayers were passaged serially by gently treating with trypsin/EDTA, and cultures were maintained in 80-mm flasks containing DMEM with 10% FBS and antibiotics. Cell cultures were grown in a humidified 5% CO2 incubator at 37uC. The strains were stored in liquid N2 until needed, and they were used between the third and seventh passage. After cells reached confluence in 6-well plates, differentiation of adipocytes was initiated by the following protocol. The culture medium were changed to serum-free DMEM supplemented with 33 mM biotin, 17 mM pantothenic acid, 10 mg/ml transferrin, 0.2 nM T3, 1 mM insulin, and 0.2 mM carbaprostaglandin, Oil Red O, and the MTT assay kit were purchased from Sigma-Aldrich, Inc.. Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, and gentamycin were purchased from Hyclone Laboratories, Inc.. Annexin V-FITC apoptosis detection kit was purchased from ” BD Biosciences.A hyaluronan enzyme-linked immunosorbent assay kit was purchased from Echelon Biosciences. Recombinant human IL-1b and TNF-a were purchased from R&D Systems. Anti-PPARc, anti-C/EBP a, anti-C/EBP b, and anti-b-actin antibodies were all obtained from Santa Cruz Biotechnology. Age GO patients: 53 46 41 55 57 44 62 Gender Duration of GO CAS Previous GO treatment Proptosis R/L Surgery performed F F F F F M M 2.2 0.8 2.3 1.4 0.9 1.5 3 3/7 2/7 1/7 0/7 1/7 3/7 3/7 GC GC GC None GC GC GC 23/24 22/19 23/23 20/23 22/24 25/25 24/19 Decompression Decompression Decompression Decompression Decompression Decompression Decompression Controls patients: 45 55 35 54 61 57 48 F F F F M M M n/a n/a n/a n/a n/a n/a n/a 0 0 0 0 0 0 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a Orbital wall fracture Orbital wall fracture Orbital wall fracture Orbital wall fracture Evisceration Evisceration Evisceration Abbreviations: CAS, clinical activity score; GC, glucocorticoids; n/a, not applicable; F, female; M, male; R/L, right or left eye. doi:10.1371/journal.pone.0026261.t001 7 October 2011 | Apigenin custom synthesis Volume 6 | Issue 10 | e26261 Effect of Quercetin in Graves’ Orbitopathy La Jolla, CA, USA). For the first 4 days, 1 mM insulin, 1 mM dexamethasone, and 0.1 mM isobutylmethylxanthine were included in the media. The differentiation was continued for 10 days, during which the media was replaced every 3 days. A PPARc agonist, rosiglitazone, was added from day 1 for further stimulation of adipogenesis. Hyaluronan ELISA Orbital preadipocyte fibroblasts were grown to confluence in 12-well plates and then incubated for indicated time periods with various concentrations of quercetin before stimulation with IL-1b or TNF-a. Supernatants from the cell cultures were collected, and hyaluronan concentrations were determined using a competitive binding hyaluronan ELISA kit according to the manufacturer’s instructions. Absorbance of reactions was measured at 405 nm, and the percentage of binding was calculated 
for each sample. The concentration of hyaluronan in the sample was determined using a standard binding curve generated with known amounts of hyaluronan. Samples were diluted 1:10 before analysis, and the average of triplicate assays was determined. Cell viability and apoptosis assays To evaluate the 10866142” effect of quercetin on preadipocyte orbital fibroblast viability, orbital fibroblasts of normal and GO patients were seeded into 24-well culture plates and treated with different concentrations of quercet