Fig. 4: ALA biosynthesis suppression mimics intestinal aging in young mice.
From: Lipoic acid functions in Paneth cells to prevent human intestinal stem cell aging
a–c Crypts from young mice were cultured for 6 days with vehicle, elesclomol, PF9366, with/without ALA. Schematic of treatment groups (a). LC-ESI-MS/MS chromatograms (b), and ALA quantification (c, n = 3 mice/group). d–i Representative images (d) (scale bars: 250 μm/50 μm) and quantitation of organoid number (e, n = 5 mice/group) and buds (f, veh: n = 13, elesclomol: n = 16, elesclomol+ALA and PF9366: n = 14, PF9366 + ALA: n = 15 organoids, all from 5 mice). Immunofluorescence for Lgr5-EGFP (green), Lysozyme (red). (g, scale bars, 50 μm, n = 6 mice/group). Quantification of Lysozyme⁺ (h, veh and PF9366 + ALA: n = 25, elesclomol: n = 35, elesclomol+ALA: n = 29, PF9366: n = 22 organoids) and Lgr5⁺ cells (i, veh, elesclomol+ALA, PF9366 + ALA: n = 19 organoids; elesclomol, PF9366: n = 18 organoids, all from 6 mice). j–r Young and old mice were injected with veh or elesclomol (j). SOX9⁺ staining (k, scale bars: 25 μm) and quantification (l, n = 10 crypts/group). Lysozyme staining (m, scale bars, 75 μm) and Paneth cell counts (n, n = 15 crypts/group, from 6 mice). Representative lysozyme granule images (o, scale bars, 10 μm; white arrowheads: normal, yellow: abnormal). Percentage of abnormal Paneth cells (p, n = 14 crypts/group). TEM of intestinal crypts (q, red dashed: Paneth cells; yellow: ISCs; yellow/red stars: normal/ abnormal granules; N, nucleus; L, lumen; M, mitochondria; scale bars: 5μm, 1μm) and mitochondrial integrity quantification (r, n = 15 cells/group from 3 mice). Data represent mean ± SD. P values: one-way ANOVA with Tukey’s test (c, e, f, h, i, l, n); f p = 5e−11 (vehicle vs elesclomol), 1e−12 (vehicle vs PF9366); h, p = 4e−10 (veh vs elesclomol), 1e−11 (veh vs PF9366); 4e−13 (elesclomol vs elesclomol+ALA), 4e−11 (PF9366 vs PF9366 + ALA); i, p = 1e−7 (veh vs elesclomol), 7e−8 (veh vs PF9366), 3e−6 (PF9366 vs PF9366 + ALA). Two-way ANOVA with Sidak’s test (p) and Tukey’s test (r); p, within D0, p = 1e–9 for young vs old; within D1–D3, p = 1e–9 for young vs old; r, within the 0–50%, p = 5e–6 (young vs young+elesclomol); within 50–100%, p = 2e–13 (young vs young+elesclomol); Source data are provided as a Source Data file.
