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then lysed on ice in 40 µL of Triton X-100 lysis buffer without
proteinase inhibitors. Lysates were cleared by centrifugation and
intracellular GSH was measured in triplicate (10 µL of sample
mixed with 50 µL of Ellman’s reagent (0.5 mM DTNB) in HEPES
buffer (0.5 M HEPES pH 7.5, 0.1 M NaCl, 0.05% Triton-X-100).
The amount of GSH was quantified by measuring the absorbance
at 405 nm in a microtiterplate reader (BioRad) and by using a
standard curve of GSH. Percentages of GSH content from treated
cells are compared to basal GSH content measured in untreated
cells.
LC-MS Analysis of Cell Lysates. Equipment: A Shimadzu
SCL-10A/Finnigan LC-QPeca with a Phenomenex Luna 5 µ C18
(150 × 4.60 mm, 5 µ) column was used, with the following gradient
(A ) 98.8% H2O, 1% MeCN, and 0.2% HCOOH; B ) 98.8%
MeCN, 1% H2O, and 0.2% HCOOH): 0 f 5 min, 0% B; 5 f 30
min, 0 f 100% B; 30 f 35 min, 100 f 0% B; 35 f 40 min, 0%
B. The flow rate was 0.4 mL/min, and detection was conducted at
m/z 414 (ionization: ESI, full MS, positive mode) or UV absorption
at 254 nm. Procedure: Cells were seeded (1.2 × 106/well) in 3
mL of medium and allowed to attach for 24 h. The supernatant
was replaced by 1.8 mL of medium containing the compound in
DMSO (final DMSO content 0.5%). For the blank, only DMSO
was used. After 24 h of stimulation at 37 °C, the supernatant was
collected and the well was washed with PBS (0.35 mL). To the
mixed supernatant and washing, 250 µL of TE was added. After
detachment of the cells, 750 µL of medium and 350 µL of 10%
HClO4 solution was added. The contents were mixed thoroughly
by vortexing for 3 min, and high-molecular material was removed
by sequential centrifugation (4000 rpm/20 min, then 14 000/15 min).
The supernatant was immediately stored at -78 °C until analysis.
Upon analysis, 20 µL was injected and the peak for 12 (at 19.8
min) was analyzed. Coinjection with authentic 12 was used to
confirm peak identity, and amounts were calculated by using the
mass areas for authentic 12.
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ing, nuclear factor-κB, and NO synthase 2 inhibition: Implications
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1992, 5, 816-822.
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two novel diterpenoid quinone methide tumor inhibitors from
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Acknowledgment. We thank Michael Bots, Saskia Hulscher,
Keren Borensztajn, Martin R. Sprick and Matthias Bickelhaupt
for valuable advice, Frans de Kanter for conducting NMR
analysis for the NAC incubations and Michaela Damsten for
assistance with LC-MS analysis.
Supporting Information Available: Purity table, graph and
additional details on computational chemistry, differences in
reactivity between acetyl ester 7a and pivaloyl ester 7b, HPLC
retention times and exact yields of the NAC incubation studies,
detailed investigations on apoptotic pathway induced by 1 and 6a,
yields of adduct 12 in HT29 cell lysates, incubation of 20 with
NAC, detailed synthetic procedures, protocols for NAC-incubation
assays and Western blots, and full analytical data for all synthesized
compounds. This material is available free of charge via the Internet
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