in for 24 h. After treatment, cells were washed, incubated with 5 mg/ml MTT solution for 4 h at 37uC, then solubilized in ice-cold isopropanol and analyzed spectrophotometrically. Absorbance of the dye was measured at 560 nm, with background subtraction at 630 nm, with a microplate reader. To evaluate the effect of quercetin on preadipocyte orbital fibroblast apoptosis, an annexin V/FITC kit was used to detect apoptotic cells. Cells were washed with isotonic phosphatebuffered saline and then incubated in serum-free DMEM in the presence of different concentrations of quercetin for 6 or 24 h, after which the apoptosis assay was performed according to the procedure recommended by the manufacturer. For flow cytometric analysis, 16104 cells were excited at 488 nm, and emission was measured at 530 and 584 nm to assess FITC and propidium iodide fluorescence, respectively. Nuclear protein extraction Orbital preadipocyte fibroblasts were plated in 100-mm dishes at 70% confluence and then incubated with various concentrations of quercetin for 24 h before stimulation with IL-1b ” for 16 h. Nuclear proteins for NF-kB western blot analysis were then isolated using a nuclear extraction kit following the manufacturer’s protocol. Briefly, cells were washed in 1 ml ice-cold PBS/phosphatase inhibitor solution, centrifuged at 3006 g for 5 min, resuspended in 500 ml ice-cold hypotonic buffer, left on ice for 15 min, vortexed, and then centrifuged at 15,0006 g for 30 s. Pelleted nuclei were ATL-962 site gently 
resuspended in 50 ml of ice-cold nuclear extraction buffer, vortexed for 15 s and then placed on ice for 15 min; resuspension and incubation of nuclei were repeated for a total of 6 times and then the 18077343” nuclear suspension was centrifuged at 15,0006 g for 5 min at 4uC. Aliquots of the supernatant that contained soluble nuclear proteins were frozen in liquid nitrogen and stored at 270uC. Semiquantitative RT-PCR Total RNA was extracted with TriZol. cDNA was synthesized from 1 mg of total RNA in a reaction containing 2 ml of a 10 mM dNTP mixture, 0.5 ml of recombinant RNasin ribonuclease inhibitor, 1 ml of AMV reverse transcriptase, reverse transcription buffer, 1 ml of Oligo15 primer. PCR was performed in a reaction containing 0.25 mM dNTP, 0.25 U Taq polymerase, 10 pmol primer pair, and 3 ml cDNA, in a thermal cycler. PCR cycling conditions for amplification of GAPDH, IL-6, and IL-8 consisted of 30 cycles of three serial segments: 94uC for 30 s, 55uC for 1 min, and 72uC for 1 min; for amplification of ICAM-1, 34 cycles of: 94uC for 30 s, 65uC for 30 s, and 72uC for 1 min; COX-2 was amplified in 35 cycles of: 93uC for 30 s, 60uC for 30 s, and 72uC for 30 s; IL-10 was amplified in 35 cycles of: 94uC for 20 s, 61uC for 20 s, and 72uC for 20 s. Primers for ICAM-1 were 59-GGC CTC AGC ACG TAC CTC TA-39 and 59-TGC TCC TTC CTC TTG GCT TA-39; for IL-6, 59-TCA ATG AGG AGA CTT GCC TG-39 and 59-GAT GAG TTG TCA TGT CCT GC-39; for IL-8, 59-TTG GCA GCC TTC CTG ATT TC-39 and 59-AAC TTC TCC ACA ACC CTC TG-39; and for COX-2, 59-GTT CCA CCC GCA GTA CAG39 and 59-GGA GCG GGA AGA ACT TGC-39; and for IL-10, 59-CTG TGA AAA CAA GAG CAA GGC-39 and 59-GAA GCT TCT GTT GGC TCCC-39. GAPDH primers were 59-GCC AAG GTC ATC CAT GAC AAC-39 and 59-GTC CAC CAC CCT GTT GCT GTA-39. Amplification bands were quantified by densitometry and normalized against corresponding GAPDH bands to control for PCR variability. Transfection and Luciferase Assays Orbital fibroblasts were transfected using the LipofectAmin200