FIG 5.

Mitochondrial reactive oxygen species are required for azaserine-induced cell death. (A) H-RasG12V-transformed STAT3 wild-type or STAT3^−/− MEFs were treated with 1 μM azaserine for 72 h in the presence or absence of 1 mM N-acetylcysteine (NAC), 1 mM glutathione ethyl ester (GSH-EE), or 10 μM MitoTempo (MitoT), as indicated, and cell viability was determined. (B) Oxygen consumption was measured in H-RasG12V-transformed STAT3 wild-type (WT) or STAT3^−/− ρ₀ MEFs using the oroboros oxygraph 2K. O₂ consumption was measured on intact cells in the absence of glucose, glutamate, and pyruvate (routine) or in the presence of 500 nM oligomycin (O), 200 nM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (F), or 20 nM antimycin A (A). (C) ρ₀ H-RasG12V-transformed STAT3 wild-type or STAT3^−/− MEFs were treated with the indicated dose of azaserine, and cell death was determined after 72 h and plotted as the means ± 1 standard deviation of the percentage of death observed in three independent experiments. Statistical significance was determined using the Student t test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.