Fig. 10: NTN1 reactivates the dampened DDR within aged HSCs. | Nature Communications

Fig. 10: NTN1 reactivates the dampened DDR within aged HSCs.

From: Restoring bone marrow niche function rejuvenates aged hematopoietic stem cells by reactivating the DNA Damage Response

Fig. 10

a–f RNA-Seq analysis of HSCs derived from young (3 month), and aged (18 month) mice treated with PBS or NTN1. a, b Comparison with an HSC aging meta-analysis (Svendsen et al)26 demonstrating alignment of young HSCs a, and aged NTN1 HSCs b, with the ‘young HSC gene signature’. c, d GSEA showing upregulation of DDR pathways in both young HSCs c and aged NTN1 HSCs d, when compared with aged PBS HSCs. e, f Heatmaps of E2F-Targets e, and DNA repair genes f, downregulated in aged HSCs, and restored after NTN1 treatment. g Representative contour plots (flow cytometry) for HSC cell-cycle analysis. h Bar graphs showing HSC cell-cycle distribution (N = 5 mice/group). i, j Assessment of mitochondrial membrane potential (MMP) by flow cytometric quantification of Tetramethyl rhodamine ethyl ester (TMRE) in HSCs of aged mice following PBS/NTN1 treatment. Bar graphs demonstrating no significant changes in % of HSCs with high MMP i, or average TMRE per HSC j, following NTN1 treatment (N = 5 mice/group). k Assessment of ROS by flow cytometric quantification of dichlorodihydrofluorescein diacetate (DCFDA) in HSCs of aged mice following PBS/NTN1 treatment (N = 5 mice/group). l, m Representative IF images for γH2AX staining within HSCs l, showing a decrease in γH2AX immunoreactivity m, following NTN1 treatment of aged mice (N = 4 mice/group). HSCs from irradiated young mice were utilized as positive controls. n, o Representative IF images of alkaline comet analysis of HSCs n, showing decrease in average Tail-moment o, and a reduction in % Tail DNA within aged HSCs following NTN1 treatment (N = 5 mice/group). p Experimental design for assessment of DNA damage and cell cycle status within HSCs following a single dose of 5-FU. q Alkaline comet analysis showing significantly reduced DNA damage within HSCs of aged mice treated with NTN1 (N = 2 mice/group). r HSC cell cycle analysis in aged mice following 5-FU treatment (N = 5 mice/group). Data is presented as the mean ± standard error of the mean (SEM). Significance determined using two-tailed unpaired t-test (pairwise comparisons), and One-Way ANOVA (multiple comparisons). *P < 0.05; **P < 0.01; ***P < 0.001. ns denotes statistically not significant.

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