Fig. 5: NR treatment decreases stress response and mitochondrial network size.

a mRNA expression levels of heat shock protein Hspa1a in aged (n = 5, biological replicates), NR-treated aged (n = 5, biological replicates) and young HSC (n = 5, biological replicates). Data are expressed as box-and-whisker plots. Box indicates 25th to 75th percentile, the line indicates the median and whiskers indicate min to max values. Statistical analysis was performed using EdgeR package, false discovery rates between two groups were determined considering the third group as background. b Quantification of Hspa1a protein levels in aged (n = 20, biological replicates), NR-treated aged (n = 10, biological replicates) and young (n = 7, biological replicates) HSPC via western blot assay. Data are mean ± s.e.m. and is a pool across three separate collection days. Statistical analysis was performed using Kruskal–Wallis, p = 0.0019 (overall) with individual groups compared using Dunn’s multiple comparisons test, p-values indicated for c. Gene set activity analysis for positive regulators of mitophagy (n = 5); the y axis shows the score as the area under the curve and has a unit of the standardised squared unit area in the range [0,1]. Data are expressed as box-and-whisker plots and is representative of a single experiment. Box indicates 25th to 75th percentile, the line indicates the median and whiskers indicate min to max values. Statistical analysis was performed using SCENIC based pipeline, employing a two-sided Student’s t-test; p-values adjusted for multiple testing using Benjamini–Hochberg (BH) correction method are indicated. d Representative Tom20 immunofluorescence staining (2D and 3D volumetric) and e 3D volumetric quantification of total cell mitochondrial network size (μm³)/HSC isolated from aged (n = 7, biological replicates), NR-treated aged (n = 5, biological replicates) and young (n = 4, biological replicates) mice, two independent experiments with 15–20 HSC quantified from each biological sample in each experimental repetition. Scale bar = 5 μm. Data are mean ± s.e.m. and statistical analysis was performed using Kruskal–Wallis, p = 0.0036 (overall) with individual groups compared using Dunn’s multiple comparisons test, p-values indicated. Determination of relative mitochondrial mass via Mitotracker green labelling in the presence of Verapamil; f representative histograms for mitotracker green level in HSPC and HSC from aged, NR-treated aged and young mice, and g relative quantification via mean fluorescence intensity for HSPC and h HSC from aged (n = 7, biological replicates), NR-treated aged (n = 8, biological replicates) and young (n = 5, biological replicates) mice. Data are mean ± s.e.m. and is representative of two independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, p = 0.0005 (overall) with individual groups compared using Tukey’s multiple comparisons test, p-values indicated for g and Kruskal–Wallis, p = 0.012 (overall) with individual groups compared using Dunn’s multiple comparisons test, p-values indicated for h. NR nicotinamide riboside, A aged, A + NR NR-treated aged animal, MTG Mitotracker green, VP verapamil, MFI mean fluorescence intensity. Source data are provided as a Source Data File.