In vitro NAD(P)H quinone dehydrogenase 1 enzyme activity
induction assay
NAD(P)H Quinone Dehydrogenase 1 (NQO1) activity was
measured as previously described
(Park et al., 2011) with slight modifications. Briefly, Hepa1c1c7 cells
were seeded in 96-well plates for 24 h and treated with the indicated
concentrations of CDDO-Me for 48 h. The cells were then lysed with
shaking for 20 min in 50 μL of cell lysis buffer (Cell Signaling Tech,
Inc., Danvers, MA) and reacted for 5 min with 200 μL of reaction buffer
(25 mM Tris-HCl; 0.07% albumin; 0.01% Tween-20; 2
U・mL-1 Glucose-6-phosphate dehydrogenase; 5 μM flavin
adenine dinucleotide; 1 μM Glucose-6-phosphate; 30 μM nicotinamide
adenine dinucleotide phosphate; 0.03% MTT; and 50 μM Menadione). After
adding 50 μL of stop solution (0.3 mM dicumarol/5 mM potassium
dihydrogenphosphate), absorbance was measured at 540 nm. The number of
cells was measured using the CellTiter-Glo assay (Promega Corp, Madison,
WI) and the absorbance value indicating NQO1 activity was corrected
using another plate seeded under the same conditions as described above.
The EC150 value, the concentration that increased the
NQO1 activity, was also calculated.