Fig. 5

Fig. 5 Mid-throughput analysis of NADP(H) dynamics in mammalian cells and mitochondria.

a Construct design for expression of NERNST and the p40phox subunit of NADPH oxidase fused to the fluorescent marker mCherry (NOX) in the cytosol and mitochondria of mammalian cells. NERNST was placed under control of the SV40 promoter in pPM014/15, and the mitochondrial targeting sequence (MTS) for mitochondrial localization in pPM026/28. L1: 30-aa linker. L2: 45-aa linker. b Spatio-temporal resolution of fluorescence R responses of the NERNST biosensor expressed in HeLa cells to successive additions of 10 mM H₂O₂ and 10 mM DTT, as shown in sequential frames (above), time-course and R values (below). Scale bars, 20 μm. Average (left) and cell-by-cell (right) analyses are shown in the lower part of the panel. Time 0 (green dots) indicates cells without treatments. Data shown are means ± SEM of 5 cells. Pseudocolor scale = R values. c Representative fluorescence images of NERNST (405 nm and 488 nm) co-expressed (bottom) or not (top) with NOX (561 nm). Scale bars, 20 μm. d NOX-mediated oxidation of the NADP(H) pool was abolished by the addition of 50 μM DPI. Data shown are means ± SD of 6-11 cells. *P ≤ 0.05, ****P ≤ 0.0001, ns, non-significant; two-way ANOVA followed by Tukey’s multiple comparisons test. e, f Kinetics of NERNST fluorescence responses in the cytosol (e) or mitochondria (f) of HeLa cells upon the addition of dimethyl sulfoxide (DMSO; control), 10 μM or 50 μM of the G6PDH inhibitor G6PDi-1, as determined with the PerkinElmer Operetta multi-well plate format confocal microscope. Data shown are means ± SEM of 7-98 cells. Representative images of cells expressing NERNST in cytosol (e) and mitochondria (f) are shown on the left. Scale bars, 10 μm. Source data, including all precise n values and exact P values, are provided as a Source Data file.