Extended Data Fig. 2: Exogenous NAD+ causes replication stress.
From: NAD+ regulates nucleotide metabolism and genomic DNA replication
a, b, NAD+ and NADH levels from metabolomics analysis relative to non-treated (NT) cells. HeLa (a) and U2OS (b) cells were treated with 2 mM NAD+ as indicated. n = 4 biological replicates, Welch’s one-way ANOVA. c, Replication fork symmetry in HeLa cells treated with 2 mM NAD+ or hydroxyurea (HU) as indicated. Symmetry of forks from Fig. 2d. d, e, Replication fork speed (d, mean ± SD) and symmetry (e) in U2OS cells treated as in c. Scored forks: non-treated (NT) = 521; 1 h NAD+ = 364; 3 h NAD+ = 493; 24 h NAD+ = 562; 1 h HU = 593. f, Immunoblot from U2OS cells treated without (NT) or with 2 mM NAD+ for the indicated hours. g, h, Relative cell number (g) and cell death (h) of HeLa cells treated without (NT) or with 2 mM NAD+ as indicated. Mean+SD, n = 12 (NT) and n = 6 (all other conditions) biological replicates, two-tailed Welch’s t-test. i, Flow cytometry analysis of DNA content in HeLa cells treated for 24 h with 2 mM NAD+ followed by 24 h in non-treated (NT) conditions. t, time. j, NAD(H) quantification in cells treated for 24 h without (NT) or with 2 mM NR. Bars represent means, n = 2 biological replicates. k, Symmetry of forks from Fig. 2f. l, QIBC analysis of γH2AX foci, EdU intensity and DNA content in U2OS cells treated without (NT) or with 2 mM of NAD+, NR or NMN as indicated. Representative from 3 independent experiments. m, Immunoblots from HeLa cells treated without (NT) or with 2 mM NAD+ or NR as indicated. n, QIBC analysis of γH2AX foci, EdU intensity and DNA content in cells treated for 24 h without (NT) or with 1 µM CD38i and/or 2 mM NAD+. o, Relative intracellular calcium changes measured with the Fluo-8 indicator in cells treated as in n. Mean+SD, n = 3 biological replicates. Replication forks were scored for two biological replicates, two-tailed Welch’s t-test. Immunoblots and flow cytometry experiments were performed twice with similar results. For boxplots in panels a, b, c, e, k, the center 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 and uncropped immunoblots are available in Source Data Files.
