**2.7 Real-time RT-PCR**

270 Rheumatoid Arthritis – Treatment

Cell viability was evaluated by the method of Mosmann (1983) using 3-(4,5-dimethyl-2-

**2.3 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)** 

MH7A cells were fixed with 4% paraformaldehyde. After inactivating endogenous peroxidase with methanol containing 0.3% H2O2, a Permeabilization Buffer (Takara Bio Inc., Otsu, Japan) was applied to cells and stood on ice for 5 min. Then, a Labeling Reaction Mixture (Takara Bio Inc.) was added and incubated in a humidified chamber at 37°C for 60 min. Reactive cells were stained with 3% methyl green and detected with a

MH7A cells were incubated in a chemiluminescence detection assay kit (Upstate, Charlottesville, Virginia, USA) and reacted with an anti-phospho-H2A.X (Ser139) followed by an anti-mouse-HRP conjugate. Phosphorylation of H2A.X at Ser139 was identified by staining with chemiluminescent HRP substrate LumiGLO, and signals were detected with a

Mitochondrial membrane potentials were measured using a DePsipherTM kit. MH7A cells were untreated and treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) for 24 h. After washing with cold phosphate-buffered saline (PBS), cells were incubated in a DePsipherTM solution at 37°C for 20 min. Then, cells were washed with 1 ml of a reaction buffer containing a stabilizer solution. The fluorescent signals were observed with a laser scanning microscopes (LSM 510, Carl Zeiss Co., Ltd, Germany) equipped with an epifluorescence device using a fluorescein long-pass filter (fluorescein and rhodamine) at an absorbance of 590 nm for red aggregations and at an absorbance of 530 nm for green

Total RNAs of MH7A cells before and after treatment with resveratrol (100 M) were purified by an acid/guanidine/thiocyanate/chloroform extraction method using a Sepasol-RNA I Super kit (Nacalai Tesque, Kyoto, Japan). After purification, total RNAs were treated with RNase free-DNase I (2 unit) at 37°C for 30 min to remove genomic DNAs, and 10 g of RNAs were resuspended in water. Then, oligo dT primers, dNTP, 5 x First Strand buffer, and SuperScript III RNase H-Reverse Transcriptase were added to the RNA solution and incubated at 65°C for 5 min followed by 60°C for 1 min, 56°C for 60 min, 58°C for 60 min, 85°C for 5 min to synthesize the first strand cDNA. Subsequently, 1 l of the reaction solution was diluted with water and mixed with 10 x PCR reaction buffer, dNTPs, MgCl2, oligonucleotide, dimethylsulfoxide [final concentration, 5% (v/v)] and 1 unit of Taq polymerase (Fermentas, St. Leon-Roth, Germany) (final volume, 20 l). RT-PCR was carried out with a Takara Thermal cycler Dice (Takara Bio Inc.) programmed as follows: the first one step, 94°C for 2 min and the ensuing 30 cycles, 94°C for 1 s, 62°C for 15 s, and 72°C for

microplate luminometer (ARVO mx/light, Perkinelmer, Waltham, MA, USA).

**2.6 Reverse transcription-polymerase chain reaction (RT-PCR)** 

thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).

**2.5 Assay of mitochondrial membrane potentials** 

**2.2 Cell viability** 

light microscope.

aggregations.

**2.4 H2A phosphorylation assay** 

**assay** 

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 City, CA, USA).


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

#### **2.8 Western blotting**

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

Resveratrol: A Candidate Drug for Treating Rheumatoid Arthritis 273

Fig. 1. Resveratrol induces apoptosis in MH7A cells. (**A**) MH7A cells were treated with resveratrol at concentrations as indicated for 24-72 h in serum-free culture medium, and cell viability was quantified with an MTT assay. In the graph, each point represents the mean (± SEM) percentage of basal levels (MTT intensities for cells untreated with resveratrol) (n=8). (**B**) Cells were treated with resveratrol at concentrations as indicated for 24 h in serum-free culture medium, and TUNEL-positive cells were counted. In the graph, each column represents the mean (± SEM) percentage of basal levels (TUNEL-positive cell numbers without resveratrol treatment) (n=3-6). *P* values, unpaired *t*-test. (**C**) Cells were treated with resveratrol at concentrations as indicated for 24 h in FBS-free culture medium, and H2A.X phosphorylation was quantified. In the graph, each column represents the mean (± SEM) ratio against basal levels (H2A.X phosphorylation without resveratrol treatment) (n=4). *P* 

values, unpaired *t*-test.

with TTBS (150 mM NaCl, 0.05% Tween20, and 20 mM Tris, pH 7.5) containing 5% BSA and subsequently reacted with an anti-cytochrome c antibody (1:400) (Chemicon, Billerica, MA, USA), followed by an HRP-conjugated goat anti-mouse IgG antibody. Immunoreactivity was detected with an ECL kit (GE Healthcare, NJ, USA) and visualized using a chemiluminescence detection system (FUJIFILM, Tokyo, Japan). Signal density was measured with Image Gauge software (FUJIFILM, Tokyo, Japan).
