Trichostatin a

For validation of the role of promoter DNA hypermethylation in transcriptional regulation of GPX7 in vitro, gastric cancer cell lines AGS and SNU1 were used. Cells were seeded at low density for 24 hours and then treated with 5 μM 5-Aza-2′ deoxycytidine (5-Aza, Sigma-Aldrich, St. Louis, MO USA) for 72 hours and/or 300 nM Trichostatin-A (TSA, Wako, Osaka, Japan) for 24 hours. Total RNA and DNA were isolated and purified by RNeasy and DNeasy Tissue kits (Qiagen), as described above. DNA methylation levels of the CpG nucleotides of the GPX7 promoter were determined by Pyrosequencing. The GPX7 mRNA expression levels were determined by qRT-PCR, as described above.

Human epithelial cell lines including intestinal HCT-8 and hepatic HepG2 were supplied by the American Type Culture Collection (ATCC; Rockville, MD, USA). Cells were maintained in RPMI 1640 medium, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin, all of which were purchased from Welgene (Daegu, Korea)], in a 5% CO₂ humidified incubator at 37 °C. Deoxynivalenol (RIS-1; purity, 97.6 ± 2.4%) was isolated from Fusarium graminearum, and anisomycin (RIS-2; purity, 98%) was isolated from Streptomyces griseolus (Sigma-Aldrich, St. Louis, MO, USA). MAPK inhibitors, including SP600125 (SP, a JNK1/2 inhibitor), SB203580 (SB, a p38 inhibitor), U0126 (a MEK1/2 inhibitor), 5-Azacytidine (5Aza, a DNA methyltransferase inhibitor), and GSK126 (an EZH2 methyltransferase inhibitor) were purchased from Sigma-Aldrich. Trichostatin A was purchased from Wako Pure Chemical Industries (Osaka, Japan).

AAV293 cells (Agilent Technologies) were cultured in 10 cm culture dish (1.25~1.5 × 10⁶ cells/dish) with 10 mL culture medium (DMEM (Sigma-Aldrich, D5796-500ML), 10% FBS (GE Life Sciences, HyClone SH30396.03), Penicillin Streptomycin (gibco, 15140-122), GlutaMax (gibco, 35050-061)). After 2~3 days, cells were passaged into T-150 flasks (2.5~3.0 × 10⁶ cells/flask) with 35 mL of culture medium. After 48~72 h, when cells reached 60~70% confluency, 25 mL of culture medium was removed, and 9 mL of fresh culture medium was added. Subsequently, plasmids were transfected via calcium phosphate co-precipitation. Briefly, three plasmids—pAAV-DJ, pHelper, plasmid including genes of interest—were added to 6 mL of 0.3 M CaCl₂. Next, 6 mL of 2x HBS (280 mM NaCl, 1.5 mM Na₂HPO₄, 50 mM Hepes) was added with thorough mixing. 3 mL of the mixed compound was applied to each flask and further cultured for 6~12 h. Next, the culture medium was replaced with 35 mL of medium containing trichostatin A (DMEM, 2% FBS, Penicillin, GlutaMax, 100 nM trichostatin A (Wako)), and further cultured for 48~96 h. Subsequently, the medium containing scraped cells was collected and underwent freeze-thaw three times. Finally, 1/10,000 volume of 250 U/µL TurboNuclease (Accelagen) was added and incubated for 30 min at 37 °C. The AAVs were purified with ViraPur Kit (Virapur).

Cells were treated either with 10 µM of 5-azacytidine (Sigma, St. Louis, MO) for 72 hours by exchanging the medium every day or with 300 ng/ml of trichostatin A (Wako, Osaka, Japan) for 24 hours. Cells were also treated with 5-azacytidine for 48 hours and then with a combination of 5-azacytidine and trichostatin A for an additional 24 hours.