Extended Data Fig. 5: Inhibiting mTORC1 activity in old mice restores intestinal regenerative capacity.

a, Organoid-forming capacity and survival of subcultured intestinal organoids treated with rapamycin. Crypts were either treated continuously for four days (2 nM) or with a two-day pulse (2 nM pulse, 10 nM pulse) followed by two days in normal medium before subculturing and quantification (n = 3). b, Regenerative growth of organoids from young and old mice treated with 2 nM rapamycin for 2 days ex vivo. Crypt number was scored six to seven days after treatment from secondary subcultures (two days after passage) (n = 5 mice per age group). Student’s paired t-test. Representative images are from subcultures on day 2. Scale bar, 100 μm. c, Weight of mice receiving daily injections of rapamycin (4 mg kg⁻¹) or vehicle (n = 5 mice per group). Daily data points represent median (circles) and interquartile range (dashed line). d, Immunoblots of pS6 from isolated crypts of vehicle (V)- or rapamycin (R)-treated young and old mice t (n = 4 mice per group). e, Organoid-forming capacity of isolated crypts from old mice treated with vehicle or rapamycin (n = 4 mice per group). f, Primary regenerative growth of organoids from old mice treated with vehicle or rapamycin (n = 4 mice per group). g, Organoid-forming capacity of young Lgr5^hi stem cells co-cultured with Paneth cells isolated from young or old mice treated with vehicle or rapamycin (n = 4 mice per group). Combinations compared to average of co-cultures with young vehicle- and old rapamycin-treated Paneth cells. h, Clonogenic growth of Lgr5^hi stem cells from young or old mice treated with vehicle or rapamycin (n = 4 mice per group); colonies quantified at day 7. i, RT–qPCR analysis of relative Wnt2b, Wnt5a, Wnt4, Wnt3 and Lgr5 expression from full jejunal samples of old mice treated with rapamycin. Values show fold change (expressed in log₂) in comparison to old vehicle treated (n values of mice analysed shown). Data are mean ± s.e.m. j, RT–qPCR analysis of relative Notum and Bst1 expression from crypts of old mice treated with rapamycin. Values show fold change (expressed in log₂) in comparison to old vehicle-treated (n = 3 mice per group). Data are mean ± s.e.m. k, Immunoblots of pS6, S6 and H3 from isolated Epcam⁺ cells of wild type (Tsc1^WT) and Tsc1 knockout (Tsc1^Δ) epithelium (n = 3 mice per group). l, Quantification of RNA-scope for Notum mRNA in wild type (Tsc1(WT)) and Tsc1 knockout (Tsc1(Δ)) ileal crypts (n = 6 mice for Tsc1(WT) and 5 mice for Tsc1(Δ)). An outlier (red) deviating >2 s.d was removed from the analysis. Representative images of crypts used in quantifications with Notum mRNA (brown) in Paneth cells (inset). m, Organoid-forming capacity of isolated crypts from Tsc1(WT and Tsc1(Δ) epithelium. Quantification was done on day 8. Y, mice between 3 and 9 months of age; O, mice over 24 months of age in all experiments. For gel source data, see Supplementary Fig. 3.

Source Data