Figure 6: PRDM16 regulates intestinal growth and maintenance by promoting FAO.

A-B) Mass spectrometry analysis of ¹³C₁₆-palmitate incorporation into TCA cycle metabolites. A) schematic. B) Relative ¹³C-labeling of the citrate/isocitrate and α-ketoglutarate pools in control (Prdm16^loxP/loxP) vs. Prdm16 mutant (R26R^CreERT2; Prdm16^loxP/loxP) crypts isolated 3d after tmx injection. ¹³C-labeling = Σ (% of isotopomer multiplied by labeled carbons in isotopomer) divided by the number of carbons in molecule. n=4-5 mice per group.

C) mRNA levels of indicated FAO genes in FACS-isolated Lgr5-GFP^Hi or Lgr5-GFP^low epithelial cells from the duodenum of Lgr5^GPP/Cre-ERT2 mice. n=3 mice.

D-G) Wildtype enteroids (in ENR medium) were treated with: (1) vehicle or etomoxir, and (2) sodium chloride (No Acetate) or Acetate (Ac). D) Brightfield images with magnified inset. E) Quantification of enteroid growth. n=32-41 enteroids. F) Quantification of enteroid budding (one or more buds). n=4 wells. G) Secondary enteroid formation from fragments of passaged enteroids. n=4 wells. Etomoxir (Eto), 50 μM in water; NaAc/NaCl, 5 mM in water. Enteroids were treated with NaAc / NaCl for 2d before addition of Eto treatment.

H-N) Control or Prdm16 mutant enteroids (in ENR medium) were treated with 5mM sodium acetate (Ac) or 5mM sodium chloride (No Acetate). H) Brightfield images with magnified insets. I) Enteroid growth. n=16-21 enteroids. J) Immunostaining of Lysozyme-positive (green) Paneth cells in control and Prdm16-mutant enteroids, 4d after inducing deletion. Enteroids were from Prdm16^loxP/loxP (control) or R26R^CreERT2; Prdm16^loxP/loxP (Mut) mice and treated with 4-OHT with additional treatment of acetate or no acetate treatment (NaCl, 5mM). E-Cadherin (red), DNA (DAPI, blue). Percentage of enteroid cells positive for Lysozyme staining are quantified in the graph. K) mRNA levels of cell type-specific marker genes in control and Prdm16 mutant enteroids at 4days post 4-OHT treatment with or without acetate treatment. n=4. L) Secondary enteroid formation from fragments of passaged enteroids. M) Enteroid budding (defined as one or more bud). n=4-6 wells. n=4 wells; NaAc/NaCl, 5 mM in water. Enteroids were pretreated with NaAc / NaCl for 2d after plating but before treatment with 4-OHT.

N) Lineage tracing of stem cell progeny in Prdm16^loxP/+ (control) or Prdm16^loxP/loxP (mutant) tdTomato reporter mice. Animals received drinking water containing either 150 mM NaAc (Ac) or NaCl (No Acetate) 2d before tmx treatment. Length of marked villus epithelial cell strips was measured for continuous strips of Tomato+ (red) epithelial cells ascending the villi at 4d post tmx treatment. n=200-291 villi measured from >=4 mice per group. tdTomato (red), DNA (DAPI, blue). O) Quantification of Cleaved caspase-3 positive cells per tdTomato positive crypt relative to the number of cleaved caspase-3 positive cells in tdTomato negative crypts. N=303-423 crypts from 3 or more mice per group.

All panels show mean ± SEM, *p<0.05, **p<0.01, ***p<0.001. Scale bars: 100 μm (D, H, J); 50 μm (N).