Fig. 5. Increased mtDNA mutation burden induces ISC decrease through NAD⁺ depletion.

a Generation of LGR5-GFP reporter mice with four categories of mtDNA mutation burden. LGR5^WT/GFP female mice were first crossed to WT/Mut** male mice, and the offspring of LGR5^WT/GFP& WT/Mut** mice were further used to generate WT/WT*&LGR5^WT/GFP, WT/Mut**&LGR5^WT/GFP and Mut/Mut***&LGR5^WT/GFP. WT/WT&LGR5^WT/GFP mice generated by crossing WT/WT male mice to female LGR5^WT/GFP female mice were used as an additional control. The figure was partly generated with the help of Servier Medical Art. b Representative images of LGR5-expressing ISCs in WT/WT&LGR5^WT/GFP, WT/WT*&LGR5^WT/GFP, WT/Mut**&LGR5^WT/GFP and Mut/Mut***&LGR5^WT/GFP male mice at 3, 8 and 12 months of age (Scale bar, 20 μm). ISC number is quantified at bottom (Data are presented as the mean ± S.D and n = 3 mice per group; two-way ANOVA test). c Representative images of LGR5-expressing ISCs in Mut/Mut***&LGR5^WT/GFP mice at 8 months of age treated with NMN or water control (Scale bar, 10 μm). ISC number is quantified on right (Data are presented as the mean ± S.D and n = 3 mice per group; unpaired two-tailed Student’s t-test). d Anti-WNT2, anti-WNT4 and anti-WNT5A IF in the intestine of WT/WT*, WT/Mut** and Mut/Mut*** mice at 8 months of age treated with NMN or water control. Relative FI is quantified on right (Scale bar, 100 μm; data are presented as the mean ± S.D and n = 3 mice per group; one-way ANOVA test). Source data are provided with this paper.