Fig. 2: Restoration of MitoCa²⁺ delays ISC aging and promotes intestinal homeostasis in aged flies.

a Representative images and quantification of cell cycle stages (shown by different color) of ISCs with indicated genotypes at different ages by the FUCCI system. ISCs (green and/or red) and nuclei (blue). Data show mean ± S.E.M (n = 858, 1504, 978 and 1100 cells, from left to right) based on two-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. Scale bar, 10 µm. b Quantification of relative lactate levels (Em 480/550 fluorescence ratios) in ISCs with indicated genotypes at different ages by Laconic, a FRET-based genetic reporter. Data show mean ± S.E.M (n = 234, 230, 256 and 226 cells, from left to right) based on two-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. c The percentage of ISCs with the indicated number of γH2AvD foci in aged flies (30 d) with the indicated genotypes was quantified. Data show mean ± S.E.M (n = 340 and 229 cells, from left to right) based on two-way ANOVA with Tukey’s multiple comparison test. *P  <  0.05 and ****P  <  0.0001. d Gut homeostasis of flies with different genotypes at different ages were monitored by the pH indicator Bromophenol Blue. “Homeostasis”, a well-defined acidic Copper cell region is flanked by basic anterior midgut and posterior midgut; “Perturbed A,” the acidic region is lost and the whole gut is basic; “Perturbed B,” the strongly acidic region is lost, while the rest of the gut also becomes less basic (weak blue). Representative images shown on the bottom. Data show mean ± S.E.M (n = 15 guts per group) based on one-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. Scale bar: 1 mm. e Food intake measured using CAFÉ assay with indicated genotypes at different ages. Data show mean ± S.E.M (n = 12 flies per group) based on one-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. f Representative images and quantification of deposit with indicated genotypes at different ages. Data show mean ± S.E.M (n = 16 fields for each group of 15 flies) based on one-way ANOVA with Tukey’s multiple comparison test. ****P  <  0.0001. Scale bar, 1 mm. Each experiment was repeated for 3 times (a–f). See Supplementary Table 1 for genotypes. Source data are provided as a Source Data file.