Fig. 4: Effects by exogenous NAD+ require mitochondrial metabolic proficiency.
From: NAD+ regulates nucleotide metabolism and genomic DNA replication
a, Metabolomics analysis in cells treated without (NT) or with 2 mM NAD+. y axis shows relative metabolite quantification, bars represent means. n = 4 biological replicates of individual samples collected from four different experiments. GLS, glutaminase. b, QIBC analysis of γH2AX foci, EdU intensity and DNA content in cells treated for 24 h without (NT) or with 2 mM NAD+, 10 µM BPTES and/or 1 mM metformin (Met). Representative experiment from three (HeLa Met) and four (all other conditions) independent experiments. c,d, Replication fork speed in HeLa (c) and U2OS (d) cells treated as in b. Scored forks in c: NT, 524; NAD+, 554; BPTES, 516; NAD+ + BPTES, 520; Met, 522; NAD+ + Met, 511. Scored forks in d: NT, 529; NAD+, 575; BPTES, 559; NAD+ + BPTES, 518; Met, 535; NAD+ + Met, 651. e,f, Replication fork speed in HeLa (e) and U2OS (f) cells treated for 24 h without (NT) or with 2 mM NAD+, 5 µM oligomycin (Oligom) and/or 1 mM 2-DG. Scored forks in e: NT, 521; NAD+, 535; Oligom, 536; NAD+ + Oligom, 537; 2-DG, 526; NAD+ + 2-DG, 541. Scored forks in f: NT, 553; NAD+, 554; Oligom, 520; NAD+ + Oligom, 515; 2-DG, 548; NAD+ + 2-DG, 571. g, Nucleotide levels from metabolomics analysis (n = 4 biological replicates of individual samples collected from four different experiments, bars represent means) and replication fork speed relative to non-treated (NT) cells from c and e. HeLa cells were treated for 24 h with 5 µM oligomycin (Oligom), 10 µM BPTES and/or 2 mM NAD+. In c–f, data are mean ± s.d., means indicated, forks were scored from two biological replicates, two-tailed Welch’s t-test. For boxplots in g, the centre line indicates the median; box limits indicate the 25th and 75th percentiles; minima and maxima of whiskers extend 1.5 times the inter-quartile range from 25th and 75th percentiles, respectively. Numerical data are available in source data files.
