Skip to main content
. Author manuscript; available in PMC: 2024 May 3.
Published in final edited form as: Nat Chem Biol. 2023 Oct 26;20(5):594–604. doi: 10.1038/s41589-023-01460-w

Figure 1. Screening of bacterial soluble transhydrogenases (STHs) for their ability to elevate NADH levels in mammalian cells.

Figure 1.

(a) The reaction catalyzed by a soluble transhydrogenase (STH). (b) Western blot analysis of HeLa cells expressing untargeted (cyto) and mitochondrially targeted (mito) bacterial STHs from E. coli, A. vinelandii, and P. putida with a C-terminal FLAG tag under doxycycline (Dox) control. Representative blots are shown. The total cellular NADH/NAD+ (c) and NADPH/NADP+ (d) ratios measured in HeLa cells expressing untargeted and mitochondrially targeted STHs from E. coli, P. putida, and A. vinelandii. (e) The lactate/pyruvate ratio measured in pyruvate-free DMEM+dFBS media, which was incubated for 3 hours with HeLa cells expressing untargeted and mitochondrially targeted STHs from E. coli and P. putida. (f) The effect of expression of untargeted and mitochondrially targeted STHs from E. coli, P. putida, and A. vinelandii on proliferation in pyruvate-free (-PYR) DMEM+dFBS. (g) The same as in (f) but in the presence of 1 μM antimycin A (ANT) and 200 μM uridine. Luciferase (LUC) and L. brevis water-forming NADH oxidase (LbNOX) expressing HeLa cells were used as controls in (b-g). Values are mean ± s.d.; n = 15, 15, 11, 12, 7, 12, 6, 6 in (c), n = 15, 15, 11, 12, 12, 12, 6, 4 in (d), n = 6, 6, 5, 6, 6, 6 in (e) biologically independent samples. Statistically significant differences were calculated by using a Welch ANOVA followed by unpaired t test. NS, no significant difference. For growth curves (f-g), error bars represent mean ± s.d.; n = 3 biologically independent samples.