Fig 1

(A) Embryos of 24 hpf were manually dechorionated, treated for 48 hours with NAD⁺ metabolites or FK-866 by bath immersion, and images obtained at 72 hpf. (B–M) Quantification of the percentage of neutrophils out of the CHT in embryos treated with NAD⁺ (0.25, 0.5 and 1 mM) (B). Representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (C). For H₂O₂ imaging, embryos were incubated in 50 μM of acetyl-pentafluorobenzene sulphonyl fluorescein solution for 1 hour. Quantification of fluorescence intensity for NAD⁺-mediated (D) and NAM-/NMN-mediated (F) induction of H₂O₂ in the zebrafish skin. Representative merge images (green and red channel) of lyz:dsRED zebrafish larvae of every group are shown (E, G). NFKB activity was determined by quantification of fluorescence intensity in embryos treated with increasing doses of FK-866 (1, 10, and 100 μM) (H). Additionally, the influence of NAD⁺ in the presence or absence of 100 μM FK-866 was assayed (I). Representative images (brightfield and green channel) of nfkb:eGFP zebrafish larvae of every group are shown (J). Neutrophil distribution in zebrafish embryos of 3 dpf treated with FK-866 (K), quantification of skin H₂O₂ production (L), and representative merge images (green and red channel) of lyz:dsRED zebrafish larvae of every group are shown (M). Each dot represents 1 individual and the mean ± SEM for each group is shown. p-Values were calculated using 1-way ANOVA and Tukey multiple range test. *p ≤ 0.05, ****p ≤ 0.0001. The data underlying this figure can be found in S1 Data. ANOVA, analysis of variance; CHT, caudal hematopoietic tissue; NAD+, nicotinamide adenine dinucleotide; NAM, nicotinamide; NMN, nicotinamide mononucleotide.