Quantification of Cellular NADPH, NADP, GSH, and ATP

The intracellular levels of NADPH and total NADP (NADPH+NADP⁺) were measured with previously described enzymatic cycling methods, with modifications^{32 (link),33 (link)}. In brief, 1.8 × 10⁶ cells were plated in 10-cm dishes; on the next day, the cells were lysed in 400 µl of extraction buffer (20 mM nicotinamide, 20 mM NaHCO₃, 100 mM Na₂CO₃) and centrifuged. For NADPH extraction, 150 µl of the supernatant was incubated at 60 °C for 30 min. Next, 160 µl of NADP-cycling buffer (100 mM Tris-HCl pH 8.0, 0.5 mM thiazolyl blue, 2 mM phenazine ethosulfate, 5 mM EDTA) containing 1.3U of G6PD was added to a 96-well plate containing 20 µl of the cell extract. After a 1-min incubation in the dark at 30 °C, 20 µl of 10 mM glucose 6-phosphate (G6P) was added to the mixture, and the change in absorbance at 570 nm was measured every 30 s for 4 min at 30 °C with a microplate reader. The concentration of NADP⁺ was calculated by subtracting[NADPH] from [total NADP]. The intracellular levels of GSH and total glutathione (GSSG + GSH) were measured with the use of enzymatic cycling methods, as described previously³⁴ . The intracellular level of ATP was measured with an ATPlite assay kit (Perkin Elmer).