Fig. 3: ACC1 deletion restricts growth and differentiation of intestinal organoids.

a–d Organoid culture of crypts isolated from ACC1^∆/∆IEC and ACC1^lox/lox mice. 4-OHT was added for 24 h after plating to induce ACC1 deletion in organoids from ACC1^∆/∆IEC mice. a Organoids were imaged at the indicated time points. Representative pictures are shown from one out of 4 independent experiments with similar results. Bar = 200 µm. Red arrows indicate crypt domains. b, d RNA was extracted from organoids at indicated time points and expression of genes was analyzed by qPCR. Data was pooled from 4 individual experiments. c Number of crypt domain formation in organoids at indicated time points. For quantification, more than 40 organoids per group were analyzed at each time point. Data shown for one representative out of 4 individual experiments. e, f Transcription levels of ISC signature genes and genes associated with secretory or absorptive IEC lineages. CPM values were derived from RNA-seq of organoids from ACC1^∆/∆IEC and ACC1^lox/lox mice at day 4 after 4-OHT treatment. RNA-seq was performed in triplicates from 3 independent experiments. g GSEA of RNA-seq for DNA replication (GO: 0006260), cell cycle (mmu04110) and chromosome segregation (GO: 0007059). h Number of secondary organoids per dissociated crypt-derived primary organoids. Data pooled from N = 3 independent experiments. i Number of organoids derived from ex vivo isolated crypts. Crypts were isolated from the upper (duo/jejunum) and lower part (ileum) of the small intestine and cultured in vitro. Data pooled from 3 independent experiments with n ≥ 3 mice per group. k Experimental set up. SorA (200 nM) or DMSO was added during organoid culture either in growth medium (upper panel) or differentiation medium (lower panel). Representative pictures of organoids are shown from two independent experiments. Bars represent 200 µm. Quantification was done by measuring the diameter of the spheroids or counting crypt domain formation. Data was pooled from 2 independent experiments, >30 organoids were analyzed for each group. l Human-derived intestinal organoids were cultured for 7 days in the presence or absence of Soraphen A (1 µM). Representative pictures shown for one out of 3 independent experiments with similar results using organoids derived from colonic resections from 3 different adult male or female patients. Bar = 200 µm. m 500 Lgr5-EGFP^high cells were sorted from the small intestine of Lgr5-EGFP-IRES-Cre^ERT2 mice and cultured either alone, or in the presence of 500 CD24^highSSC^high paneth cells sorted from ACC1^∆/∆IEC mice. Representative images of organoids are shown from two independent experiments, bar = 200 µm. For quantification, diameter of organoids was measured. Data was pooled from 2 independent experiments, ≥13 organoids were analyzed for each group. Statistical significance was analyzed using unpaired two-tailed Student’s t-test (h, i, k), Benjamini–Hochberg procedure (e, f) or One-way ANOVA with Tukey’s multiple comparison test (b, c, d, m) with *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001. Exact p values provided as Source Data. Bar graphs represent mean and error bars indicate SD. Boxes in boxplots denote 25th to 75th percentiles with whiskers representing min-max, and the central line the median. Source data are provided as a Source Data file.