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B. C. Das et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4204–4206
18. Wang, G. L.; Jiang, B. H.; Rue, E. A.; Semenza, G. L. Proc. Natl. Acad. Sci. U.S.A.
Acknowledgments
1995, 92, 5510.
19. (a) Cell culture and cytotoxicity assays: HepG2 cells (passage 2 from frozen
stock) were cultured in RPMI 1640 containing 10% FBS. The tests were
performed in 96-well plates with stock cells split equally into duplicate
plates—one for normoxic and the other for hypoxic conditions. The drug(s) or
vehicle (0.1% DMSO) was applied to the cells in log-exponential growth phase
under normoxic conditions (5% CO2; 37 °C) for 48 h or under normoxic
conditions (22 h) followed by 2 h hypoxic conditions (1% O2, 5% CO2, 94% N2 at
37 °C) then followed by reoxygenation for 24 h. For the hypoxia experiments,
we used a glove chamber with two-way pressure value and one-way N2 inflow.
Hypoxia conditions were met using methylene blue dye indicator (i.e., 0.015%
methylene blue, 6% dextrose, 6 mM NaOH) which correlated with the O2
pressure in the glove box when measured continuously with a Clark-type
electrode. At 48 h, a tetrazolium component (MTT) assay (Promega, Madison,
WI; Cell Titer 96 Non-Radioactive Cell Proliferation Assay) was performed
following an established protocol in the laboratory.
B.C.D. acknowledges Professor E.R. Stanley, Professor Susan B.
Horwitz and Professor M. Donald Blaufox for their support and
encouragement and AECOM start up funding. All authors are thank-
ful to Dr. S. M. Mahalingam for his help in manuscript preparation.
Supplementary data
Supplementary data (biological and chemical experimental pro-
cedures, spectroscopic data, and NMR spectra for all new com-
pounds) associated with this article can be found, in the online
(b) HepG2 clonogenic assays: 3.5 Â 103 per well were seeded in 6-well plates
using RPMI1640 and 10% FBS. After 24 h of growth at 37 °C, the cells were
exposed to vehicle or drug(s). After 10 days incubation with drug(s) or control,
the wells were washed with PBS twice, then fixed in 10% acetic acid for 10 min,
then stained with crystal violet for 10 min, and finally rinsed with distilled
References and notes
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TM
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