Fig. 3: Metabolic and epigenetic divergence in intestinal progenitors.
From: Metabolic adaptations direct cell fate during tissue regeneration
a, Immunofluorescence in C57Bl/6 progenitor-enriched organoids showing TET and 5hmC expression. b, Steady-state levels of αKG and αKG/succinate ratio by LC–MS in ISC-enriched and progenitor-enriched organoids. Each dot represents a replicate, generated by isolating and pooling crypts from five mice and plating each in triplicate in a separate well. Data are representative of two independent experiments (n = 10 mice). c, Oxygen consumption rate (OCR) in organoids derived from TRE-shRenCag-rtTA3 or TRE-shOgdhCag-rtTA3 mice (n = 4 for shRen and n = 4 for shOgdh). Olig., oligomycin; R+A, rotenone and antimycin. d, ATP production in TRE-shRenCag-rtTA3 or TRE-shOgdhCag-rtTA3 mice as measured on a Seahorse instrument (n = 4). Each dot represents one mouse. e, Succinate, fumarate, aconitate and citrate shown as fractional labelling with 13C5-glutamine (n = 3 per condition) in organoids from TRE-shOgdhCag-rtTA3 mice, with or without doxycycline treatment for 72 h. Each dot represents a replicate, generated as explained in b. Data are representative of two independent experiments (n = 10). f, Immunofluorescence for HNF4α and 5hmC in crypts in tissue sections from C57Bl/6 mice. Dashed lines outline 5hmC+HNF4α− cells. Scale bar, 20 μm. g, Lack of colocalization between HNF4α and 5hmC in tissues derived from f. Each dot represents one mouse (n = 5). h, 5hmC and HNF4α expression in individual cells from tissue sections obtained from f. Each dot represents one cell (n = 5 mice). i,j, Expression of Turbo–GFP reporter in the indicated intestinal lineages (i) and GFP/luciferase ratio in enterocytes at 72 h (j) electroporated with a reporter construct containing either wild-type or Hnf4a-mutant binding sites in the Ogdh promoter (n = 3 mice; 8 wells per mouse). RFI, relative fluorescence intensity; RLU, relative luminescence units. The box represents the interquartile range (IQR) with the median as a central line. Whiskers extend to 1.5 × IQR beyond Q1 and Q3. This figure is adapted from our published patent (WO2024229094A1)50. Data are mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test in b,g, two-tailed t-test in d,e,j and two-way ANOVA followed by Tukey’s HSD test in c. Asterisks indicate statistical significance (**P < 0.01). Scale bars, 10 μm (a,i), 50 μm (f).
