Resveratrol

Protease and phosphatase inhibitors were purchased from Roche Diagnostics (Basel, Switzerland). Resveratrol and N-acetylcysteine (NAC) were procured from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Acetoxy-forms of HCAs (PhIP: molecular weight 286.3; MeIQx: molecular weight 275.2) were synthesized (NARD Institute, Amagasaki, Hyogo, Japan). FICZ is a tryptophan photoproduct (Enzo Life Sciences, NY, USA). GW6471 PPARα inhibitor was purchased from Wako (Osaka, Japan). Neomycin (Wako) and hygromycin (Hygro, Sigma, MO, USA) were used after checking the cytotoxic effects in each cell line. Antibodies against PPARα (Abcam, Tokyo, Japan), sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), sirtuin 6 (SIRT6), sirtuin 7 (SIRT7), p38, phosphorylated p38, CREB, phosphorylated CREB, and SNF2H (Cell Signal Technology, Tokyo, Japan), H2AX (Millipore), enhanced green fluorescent protein (MBL, Nagoya, Japan), Anti-FLAG Tag M2 antibody (Sigma MO, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA) were used as primary antibodies. A rabbit polyclonal antibody against human ORF1 was generated using the peptide MGKKQNRKTGNSKTQSAC as an immunogen (Medical and Biological Laboratories)^{14 (link)}. Anti-mouse and anti-rabbit secondary IgG conjugated to horseradish peroxidase were obtained from DAKO Japan (Tokyo, Japan).

Crushed grape powder (including pericarp, seed, flesh, and grape stem) (Zero Co. Ltd., Chiba, Japan) was dissolved in 70% ethanol, macerated for 7 days, and centrifuged at 3000 rpm for 20 min. The supernatant was filtered through a 0.22 μm membrane and completely dried. The resulting grape extract was dissolved in DMSO and stored at –80 °C. Resveratrol (Wako, Osaka, Japan) was also dissolved in DMSO at 100 mM and stored at −80 °C. A UPLC system (UPLC–SYNAPT G2-S HDMS) from Waters Co. (MA, USA) was used to roughly calculate the Resveratrol content in the grape extract (Figure S3). HPLC separation was achieved on a 2.1 × 150 mm column (Waters Co., MA, USA). The mobile phases consisted of (A) water and (B) methanol. The separation was carried out at room temperature with a 0.2 mL/min flow rate, under the following conditions: 0–30 min, 0–50% B, 30–50 min, 50–95 % B, 50–55 min, 95 % B isocratic, 55–60 min, 100–0% B, followed by a 5 min re-equilibration time of the column. Calculated by comparing the peak area at the same position, the grape extract contained 7% Resveratrol, so the concentration of Resveratrol in 50 μg/mL (100 μg/ml) grape extract was determined to be about 15 μM (30 μM).

We examined the inhibitory activities of food constituents in cell proliferation assays. Test compounds used in the assays were as follows: curcumin (Wako or Sigma-Aldrich), zerumbone (Wako), genistein (Sigma-Aldrich), chalcone (Wako), corosolic acid (Wako), carnosol (Wako), ursolic acid (Wako), epigallocatechin (Sigma-Aldrich), isoflavone (Wako), apigenin (Wako), chrysin (Wako), flavanone (Wako), resveratrol (Wako), flavone (Wako), quercetin (Wako), daidzein (Wako), and clodronate (Sigma-Aldrich). These compounds were firstly dissolved with dimethyl sulfoxide (DMSO; Wako) and then appropriately diluted with RPMI1640 + 10% FCS to apply the assays. The final concentration of DMSO was adjusted not to exceed 1% at most in cell cultures.