Intracellular oxidation of pyridine nucleotides induces a conformational change in NQO1.

(A) 16HBE cells were treated with DMSO or β-lapachone (10μM) for 2 h after which NQO1 was immunoprecipitated using anti-NQO1 antibodies. (B) Human cell lines (16HBE, ARPE19, TrHBMEC) were treated with β-lapachone (10μM) for the indicated times after which NQO1 was immunoprecipitated using antibodies that recognize the C-terminal domain of NQO1. (C) Intracellular levels of NADH and NAD⁺ were measured by mass spectrometry in 16HBE cells treated with β-lapachone (10μM) for 2 h in the absence and presence of the PARP inhibitor olaparib (1μM). Results are the mean ± standard deviation, n = 3. (D) Immunoprecipitation of NQO1 from 16HBE cells treated with β-lapachone (10μM) in the absence and presence of olaparib (1μM) for 2 h. Reaction conditions are described in Materials and methods.