Fig. 4.

A reduced cytosolic NADH/NAD⁺ redox state suppresses intrinsic apoptosis induced by oxidative stress. a HepG2 cells were incubated in medium with L/P ratios of 1 and 250 for 1 h prior to addition of Raptinal (or vehicle, 0.1% DMSO). The oxidation rate of DHE was monitored fluorometrically. Rates of DHE oxidation were normalized to vehicle-treated cells under oxidized redox clamp (L/P = 1) set to 100%. Data are represented as mean ± SD and are representative of two independent experiments. b HepG2 cells were pre-incubated for 1 h in media containing different L/P ratios. Following pre-incubation, vehicle (0.1% DMSO) or 10 µM Raptinal was added for 2 h. Cytosolic fractions were separated from non-cytosolic fractions (containing mitochondria). To assess cytochrome c release from mitochondria, equal volumes of cytosolic and non-cytosolic fractions were immunoblotted for cytochrome c. Shown is a representative experiment of N = 3. c Treatment as in (b) but whole cell lysates were prepared and immunoblotted for cleaved caspase-3 and its substrate, PARP-1. Shown is a representative experiment of 3 independent experiments (N = 3). d HepG2 cells were pre-incubated for 1 h in media with different L/P ratios after which 40 ng/mL TRAIL was added for 4 h. Caspase 3/7 activity was then measured. Data are normalized to vehicle condition under L/P = 1 and presented as mean ± SD of a representative experiment (N = 3). Statistical analysis (a, d): two-way ANOVA with multiple comparison (Tukey) with ****p < 0.0001, ns not significant