Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P) H: quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (E-GSH) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (beta-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors. Immediate oxidation of EGSH in both the cytosol and mitochondrial matrix was observed in response to DNQ and b-lap. The DNQ-induced cytosolic oxidation was fully prevented with NQO1 inhibition, whereas mitochondrial oxidation in A549 was NQO1independent in contrast to MIA-PaCa-2 cells. However, at pharmacologic concentrations of b-lap both quinone-based substrates directly oxidized the redox probe, a possible sign of off-target reactivity with cellular thiols. Together, these data provide new evidence that DNQ's direct and discerning NQO1 substrate specificity underlies its pharmacologic potency, while b-lap elicits off-target responses at its effective doses. (C) 2016 Elsevier Inc. All rights reserved.