Figure 1. WRN is essential in maintaining metabolic, mitochondrial, and other longevity-related pathways as well as in inhibiting senescence in mesenchymal stem cells. (A, B) Gene-set-enrichment analysis demonstrates upregulated and downregulated signaling pathways (KEGG pathways) in WT and WRN−/− MSCs with/without 1 mM NR treatment for 24 h. The KEGG terms were ranked on the basis of enrichment scores. The upregulated KEGG pathways and downregulated KEGG pathways are summarised separately. (C) Heat map data showing fpkm changes of significantly up- or down-regulated genes (DEGs) related to NAD+ metabolism in the comparisons shown (list of target genes is found in Supplementary Table 1; full list of fpkm values is found in Supplementary File 1). WRN−/− (Veh) clusters alone, whereas WRN−/− (NR) and WT (Veh) cluster together. (D) Correlation matrix of fpkm values showing the correlation between expression of NAD+ related DEGs and proliferation/senescence related markers. (E–G) Senescence evaluation by senescence associated β-Galactosidase (SA-β-Gal) staining of MSCs derived from control (WT) and WS patients (WS). SA-β-Gal positive cells decreased with 11–18 days of 1 mM NR treatment in WRN−/− MSCs (Student’s t-test, p-values = 0.0097 and 0.0156, respectively) (F, G), but not in WT MSCs (Student’s t-test, p-value = 0.2029) (E). SA-β-Gal positive MSCs relative to total number of cells were quantified from two biological experiments per cell line. (H) SA-β-Gal staining of primary fibroblasts from healthy control donors (WT) and WS patients (WS). SA-β-Gal staining was increased in WS patient derived fibroblasts compared to healthy controls (WT) (Two-way ANOVA, Tukey’s multiple comparisons test, p-value = 0.0036). SA-β-Gal staining decreased with 10 days 1 mM NR treatment in WS patient derived primary fibroblasts (Two-way ANOVA, Tukey’s multiple comparisons test, p-value = 0.0115) (F, G), but not in WT MSCs (Two-way ANOVA, Tukey’s multiple comparisons test, p-value = 0.9930) (Senescence evaluation by Spider β-Gal (Dojindo) staining of primary fibroblasts from healthy donors (WT) or WS patients (WS) without or with 1 mM NR for 10 days prior to staining are found in Supplementary Figure 1). (I) Staining of HMGB1 in primary fibroblasts from healthy donors (WT) or WS patients (WS) without or with 1 mM NR treatment for 10 days prior to staining. The number of cells with nuclear HMGB1 only relative to the total number of cells is shown in the figure from three biological repeats from two WT cell lines and two WS cell line. The percentage of cells with nuclear HMGB1 was decreased in WS-derived primary fibroblasts compared to WT (Two-way ANOVA, Tukey’s multiple comparisons test, p-value < 0.0001). Supporting the SA-β-Gal staining, 10 days 1 mM NR treatment increased the proportion of WS-derived fibroblasts with nuclear HMGB1 staining compared to vehicle treated cells significantly (Two-way ANOVA, Tukey’s multiple comparisons test, p-value = 0.0006), indicating decreased senescence with NR treatment.