ARTICLE;
COMPARATIVE MOLECULAR FIELD ANALYSIS;
COMPARATIVE MOLECULAR SIMILARITY INDICES ANALYSIS;
DRUG ACTIVITY;
DRUG STRUCTURE;
ENZYME INHIBITION;
TRANSCRIPTION INITIATION;
D. Subramaniam, P. Giridharan, N. Murmu, N.P. Shankaranarayanan, R. May, C.W. Houchen, R.P. Ramanujam, A. Balakrishnan, R.A. Vishwakarma, and S. Anant Cancer Res. 68 2008 8573
E. Pikarsky, R.M. Porat, I. Stein, R. Abramovitch, S. Amit, S. Kasem, E. Gutkovich-Pyest, S. Urieli-Shoval, E. Galun, and Y. Ben-Neriah Nature 431 2004 461
HCT116 human colon cancer cells (American Type Culture Collection) were maintained in DMEM medium supplemented with 10% fetal bovine serum (Hyclone). NF-κB-dependent transcription was measured with a cis-acting reporter assay system. HCT116 cells were seeded onto 12-well plates and transfected with 0.5 μg of pNF-κB-Luc, which contains five repeats of NF-κB binding sites (Stratagene), using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. To monitor transfection efficiency, 50 ng of the pRL-null plasmid encoding renilla luciferase (Promega) was included in all samples. At 72 h post-transfection, levels of firefly and renilla luciferase activity were measured sequentially from a single sample of cells, either untreated or treated with 10 ng/mL TNFα in the absence or presence of the test compound for 12 h, using the Dual-Glo™ Luciferase Assay System (Promega). The normalized level of luciferase activity in the TNFα-only treated sample was designated as 1.22.
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63349085877
S. Lee, Y. Woo, S.Y. Shin, Y.H. Lee, and Y. Lim Bioorg. Med. Chem. Lett. 19 2009 2116
Data not shown in the text can be found in the Supplementary data
Data not shown in the text can be found in the Supplementary data.
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77957861724
The field contributions for the analysis of CoMFA and CoMSIA contour maps were determined based on matching the physicochemical properties of the template showing the best biological activity with the field descriptors used
The field contributions for the analysis of CoMFA and CoMSIA contour maps were determined based on matching the physicochemical properties of the template showing the best biological activity with the field descriptors used.
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77957868764
note
HCT116 cells were treated with 5,7,3′,4′- tetramethoxyisoflavone for 30 min, followed by the addition of TNFα for 30 min. Cells were harvested and the nuclear fractions were collected. Total nuclear lysates (20 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose filters. The filters were blocked with 5% non-fat dry milk and were probed with primary antibodies, anti-p65 NF-κB antibody (Santa Cruz Biotechnology), and anti-lamin B antibody (Santa Cruz Biotechnology). Lamin B was used as a marker for the nuclear fraction. Positive reactions were visualized with an enhanced chemiluminescence plus Western blotting detection system (Amersham Bioscience).
27
77957873412
note
HCT116 cells were treated with 5,7,3′,4′- tetramethoxyisoflavone for 30 min, followed by addition of TNFα for 18 h. Total RNA was extracted using a Trizol RNA extraction kit (Invitrogen). The first-strand cDNA was synthesized from 500 ng of total RNA using an iScript cDNA synthesis kit (Bio-Rad). For RT-PCR analyses, gene-specific primers for CXCL1 (forward, 5′-atggcccgcgctgctctctcc-3′; reverse, 5′- gttggatttgtcactgttcag-3′), XIAP (forward, 5′-acaccatatacccgaggaac- 3′; reverse, 5′-cttgcatactgtctttctgagc-3′), CIAP2 (forward, 5′-cctgtggttaaatctgccttg-3′; reverse, 5′- caattcggcaccataactctg-3′), and GAPDH (forward, 5′- tcgacagtcagccgcatcttc-3′; reverse, 5′-cgcccaatacgaccacctccg- 3′) were used. The PCR conditions for all primers were as follows: hold for 5 min at 94 °C, followed by 30 cycles of denaturation at 94 °C (30 s), annealing at 55 °C (30 s), and elongation at 72 °C (1 min). The amplified products were separated by electrophoresis on a 1% agarose gel.
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C.H. Lee, Y.T. Jeon, S.H. Kim, and Y.S. Song BioFactors 29 2007 19
