**2.1 Cell culture**

MH7A human rheumatoid arthritis synovial cells were obtained from the Riken cell bank (Ibaraki, Japan). Cells were cultured in a culture medium; RPMI-1640 (Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml), in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

Resveratrol: A Candidate Drug for Treating Rheumatoid Arthritis 271

30 s using primers shown in Table 1. PCR products were stained with ethidium bromide

Bad CTGGGGCTGTGGAGATCCGGAGTCGCC TCACTGGGAGGGGGCGGAGCTTCCCC 320 Bak GAGCCCATTCCCACCATTCTACCT AGAGAGGAAGGGAGAGAACTGAGAGGAC 381 Bax GGGAGACACCTGAGCTGACC GGACTCCAGCCACAAAGATGG 404 Bcl-2 GAACTGGGGGAGGATTGTGGCC TCGACGTTTTGCCTGAAGACTGTTAA 486 Bcl-XL AGGGAGGCAGGCGACGAGTTT TGAAGAGTGAGCCCAGCAGAACCA 421 Sirtuin 1 ATTACTGAAAAACCTCCACGAACACAAAA GCCTACTAATCTGCTCCTTTGCCACTCT 379

Total RNA was isolated from MH7A cells with Sepasol RNA I super kit (Nacalai Tesque, Kyoto, Japan). Total RNA (5 g) was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Tokyo, Japan), and quantitative realtime RT-PCR was performed with Applied Biosystems 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the SYBR Green realtime Master Mix (TOYOBO, Osaka, Japan) protocol. Primers used for real-time RT-PCR are shown in Table 2. The PCR cycling conditions were 95C for 4 min, followed by 40 cycles at 95C for 15 s and 62C for 15 s and 72C for 45 s. A standard curve was made by amplifying 0.5, 1, 2, 4, and 8 μl of the GAPDH mRNA diluted at 1:250. The mRNA quantity for each target gene was calculated from the standard curve using an SDS 2.1 Software (Applied Biosystems, Foster

name Sense primer Anti-sense primer Base

AIF TCACAAAGACACTGCGATTCAAACAGT GTTGCTGAGGTATTCGGGGAGGAT 491 FOXO-1 TATGGACACTTTGCGTTTCTTATTTAGGATAAC GGCAGAAGGGAGAATGAGATGAAGTATG 366 FOXO-3 TGGATGCTGATGGGTTGGATTTTAA TGGTGCTGAGGGGTGCTGTCC 415 P21 ACCGAGTGGGGGCATCATCAA GGATGGGGTGGATGAGGAAGGTC 534 P27 CGTGGCTCGTCGGGGTCTGT CCCTTCTCCACCTCTTGCCACTC 393 P53 GCCATCTACAAGCAGTCACAGCACAT GGCACAAACACGCACCTCAAAGC 348 GAPDH GACTTCAACAGCGACACCCACTCC AGGTCCACCACCCTGTTGCTGTAG 128

After treatment with resveratrol (100 M) for 6-24 h, MH7A cells were harvested and centrifuged at 600 x g for 10 min. After washing-out with 1 ml of PBS, the pellet was resuspended in 50 l of buffer A (20 mM Hepes, 10 mM KCl, 1.5 mM MgCI2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 250 mM sucrose, pH 7.5) and homogenized. The lysate was centrifuged at 1000 x g for 10 min and the supernatant was further centrifuged at 10000 x g for 1 h. The pellet and supernatant were regarded as the mitochondria- and cytosol-enriched fractions, respectively. Each fraction was loaded on 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Blotting membranes were blocked

Sense primer Anti-sense primer Base

pair

pair

and visualized by 2% agarose electrophoresis.

Table 1. Primers used for RT-PCR

Table 2. Primers used for real-time RT-PCR

**2.7 Real-time RT-PCR** 

City, CA, USA).

**2.8 Western blotting** 

Gene

Gene name
