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Thienotriazolopyrimidine Derivatives Inhibit STAT3 Activation Induced by IL-6
Thienotriazolopyrimidine Derivatives Inhibit STAT3 Activation Induced by IL-6
Bulletin of the Korean Chemical Society. 2014. Aug, 35(8): 2570-2572
Copyright © 2014, Korea Chemical Society
  • Received : March 06, 2014
  • Accepted : April 17, 2014
  • Published : August 20, 2014
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About the Authors
Seung Woong Lee
Hyun-Mee Oh
Eco-friendly Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup-si 580-185, Korea
Mun-Chual Rho
Eco-friendly Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup-si 580-185, Korea
Yang-Heon Song

Abstract
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Experimental Section
Reagents and Chemicals. Recombinant human IL-6 was purchased from R&D systems (Minneapolis, MN, USA). All reagents including genistein were obtained from Sigma-Aldrich Ltd (St Louis, MO, USA).
Cell Line and Cell Culture. Human hepatoma Hep3B cells were obtained from American Type Culture Collection (ATCC No. HB-8064, Rockville, MD) and were maintained in a DMEM medium, supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 mg/mL streptomycin, at 37 ℃ in a 5% CO 2 incubator. All cell culture reagents were obtained from GibcoBRL (Life Technologies, Cergy-Pontoise, France).
Establishment of Stable Cell Line Expressing pStat3- Luc. Hep3B cells were cotransfected with pStat3-Luc encoding the Stat3 binding site and pcDNA3.1 (+) carrying a hygromycin selection marker (Clontech laboratories, Palo Alto, CA) by using lipofectamin plus (Invitrogen, Carlsbad, CA, USA). Two days after the transfection, cells that stably expressed luciferase were selected by hygromycin treatment (100 µg/mL) and stable clones were expanded. Expression of luciferase in the clones stably expressing pStat3-Luc was confirmed by luciferase assays.
Luciferase Assay. Hep3B cells stably expressing pStat3-Luc were seeded on to 96-well culture plates at 2 × 10 4 cells/well. After 24 h, cells were performed with starvation for 12 h, and then treated with IL-6 (10 ng/mL) with or without compounds for 12 h. Luciferase assay was performed with the kit from Promega according to the manufacturer’s protocol.
Western Blot Analysis. Total proteins were prepared from cells and subjected to Western blot with primary rabbit anti-phospho Stat3 (Tyr705) IgG, anti-total Stat3 IgG (1:1000), and secondary antibody (1:2000).
Cell Viability. Hep3B cells were seeded at a plating density of 2 × 10 4 cells/well and cultured for 24 h to allow them to adhere to the plate. After 24 h, the culture medium changed to the serum free medium supplemented with the samples indicated dose. Following the culture with sample for 48 h, MTT (0.5 mg/mL) was added, and after 4 h of incubation at 37 ℃, 200 µL of DMSO was added into each well. The absorbance of the samples at 540 nm was measured against a background control by using a 96-well plate reader. The percentage of viable cells under each treatment condition was determined according to the negative control.
Acknowledgements
This work was supported by the Korea Research Foundation (project number 2010-0021038).
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