Epigallocatechin 3 gallate egcg

Anti-malarial drugs, pyrimethamine (PYR, MW = 249) and dihydroartemisinin (DHA, MW = 284) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). DFO (desferrioxamine mesylate or Desferal^®, MW = 657) was from the Novartis Pharma, Basel, Switzerland, Chiang Mai. DFP (GPO-L-One^® or L1, MW = 139) and DFX (Exjade^®, MW = 373) were kindly supplied by the Institute of Research and Development, Government Pharmaceutical Organization, Bangkok, Thailand. SYBR Green I nucleic acid gel-stain (10,000× concentrate in DMSO, Cat. No. S9430) was purchased from Invitrogen, Molecular Probes (Eugene, OR, USA). Calcein acetoxymethyl (CA-AM) was purchased from Invitrogen^® Corporation (CA, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) and epigallocatechin 3-gallate (EGCG, MW = 458) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). SYTO^® 61 red fluorescent nucleic acid dye (Cat. No. S11343) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and prepared fresh prior to each experiment by making a 1 mM stock solution in PBS. (-)-epigallocatechin-3-gallate (EGCG) and (-)-gallocatechin gallate were purchased from Sigma-Aldrich and were prepared in dimethyl sulfoxide. Pro-EGCG was prepared as we previously described, and a 50 mM solution was prepared in DMSO for in vitro studies [23 (link)].

The epigallocatechin-3-gallate (EGCG) was purchased from Sigma-Aldrich (St Louis, MI). The two synthetic EGCG derivatives: 1,5-Bis [(3,4,5-trihydroxybenzoyl) oxy] naphthalene (compound 23) and 4,4′-Bis [(3,4,5-trihydroxybenzoyl) oxy]-1,1′-biphenyl (compound 30) were synthesized as previously described [21 (link)]. The Dc protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). The Total RNA isolation nucleo-spin RNA II kit was from Macherey-Nagel (Düren, Germany) and the StratraScript First Strand cDNA Synthesis System was purchased from Stratagene (Santa Clara, CA). The EC Western Blotting Detection Reagent was from Santa Cruz Biotechnology (Santa Cruz, CA) and the Hybond-C Extra Nitrocellulose membranes were purchased from Amersham (Little Chalfont, UK). All other chemicals were obtained from Sigma-Aldrich. Finally, the 6–0 poly-glycolic acid synthetic absorbable suture and 5–0 nylon suture were purchased from Suturas Aragó (Barcelona, Spain).

Peripheral blood CD4⁺CD25⁺ Tregs were collected and isolated from one healthy volunteer by magnetic cell separation using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to our previous descriptions [14 (link)]. Isolated CD4+CD25+ Tregs were cultured and stimulated based on previously reported protocols with minor modifications [16 (link), 17 (link)]. Isolated CD4+CD25+ Tregs were cultured at a density of 2.5 × 10⁵ cells per well in a 24-well culture plate supplemented with soluble anti-CD3 (1 mg/ml), IL-2 (100 U/ml), and 50 μg/ml fresh oxidized low-density lipoprotein (ox-LDL) (Peking Union-Biology, Beijing, China). In inhibition experiments, Tregs were stimulated with inhibitors of DNA methyltransferases (5-aza-2′-deoxycytidine, AZA [10 μM] and epigallocatechin-3-gallate, EGCG [40 μM]) (Sigma-Aldrich, St. Louis, MO, USA) and were transduced with DNMT3b shRNA (multiplicity of infection =25), respectively. Tregs were cultured for 72 hours in an atmosphere of 5% CO2 at 37°C. The experiments were repeated three times and each was from one healthy volunteer. The healthy volunteer gave informed consent to participate in this study, which was approved by the Ethics Committee of Xi'an Jiaotong University.

Fresh pork logissimus thoracis within 24 h post mortem was purchased from a local supermarket (Yangling, China). Epigallocatechin-3-gallate (EGCG) and amylopectin (extracted from corn) were purchased from Sigma Chemical (St. Louis, MO, USA) and Macklin Biochemical Co., Ltd. (Shanghai, China). All chemicals used in this study were of analytical reagent grade.

Xanthohumol ((2E)-1-[2,4-dihydroxy-6-methoxy-3-(3-methyl-2-buten-1-yl)phenyl]-3-(4-hydroxyphenyl)-2-propen-1-one) was purchased from Alexis Biochemicals (San Diego, CA, USA). Epigallocatechin-3-gallate (EGCG) was purchased by Sigma Aldrich (Sigma-Aldrich, St Louis, MO, USA). STO-609 was purchased from Sigma-Aldrich (St Louis, MO, USA) and Compound C from CalbioChem (Darmstadt, Germany). The ON-TARGET plus SMART pool siRNAS targeting AMPK α1 (PRKAA1 L-0502700) and ON-TARGET plus Nontargeting Pool negative control siRNA (D-001810) were purchased from Dharmacon (Lafayette, CO). Lipofectamine RNAiMAX (catalog number 13778-075) was purchased from Invitrogen (Eugene, OR).

Epigallocatechin-3-gallate (EGCG) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in distilled water as a stock solution. PANC-1 and HEK293 cells were seeded into 35-mm-diameter culture dishes (1 × 10⁵ cells/dish) and then treated with various concentrations of EGCG (0, 10, 20, 30, 40, and 50 μM). After the cell viability assay, only 10 and 20 μM of EGCG were used in the following experiments.