Fig. 3: Differentiation with small molecule AS1842856 induces CHGA expression.

a Representative light microscopy of enteroids (whole well) grown in growth media for 2 days followed by 12 days in differentiation media with AS1842856 (AS). Culture schematic located above panel. Scale bar = 1 mm. b qPCR analysis of enteroendocrine markers of enteroids grown in G14, G2D12 or AS compared to whole duodenal mucosa and normalized to 18S. Dotted line denotes expression level in whole duodenal mucosa. Representative experiment showing n = 3 wells from each condition from a single enteroid line. CHGA = chromogranin A, PAX4 = paired box 4, PDX1 = pancreatic and duodenal homeobox 1, NEUROD1 = neuronal differentiation 1, NEUROG3 = neurogenin 3, CCK = cholecystokinin, SST = somatostatin, GIP = glucose-dependent insulinotropic peptide, ND = not detectable in one or more samples. *p = 0.038 (NEUROG3), 0.0130 (CCK); ***p = 0.0004 (PDX1, G14 to AS), 0.0009 (PDX1, G2D12 to AS); ****p < 0.0001. c Representative immunofluorescence staining of CHGA (magenta) in enteroids (whole well) treated with G14, G2D12 and AS. Boxed portion magnified in lower right corner. DNA (4′,6-diamidino-2-phenylindole (DAPI), blue). Scale bars = 1 mm and 50 µm (boxed portion). d Percentage of enteroids with positive CHGA staining in G14, G2D12 and AS treatments. Table above graph shows the total number of enteroids examined per condition. Average results are from three separate experiments from three different enteroid lines or passages. ****p < 0.0001. e Left three panels: Representative flow cytometry plots of CHGA + cells from enteroids grown in G14, G2D12 or AS. Right panel: Percentage of CHGA + cells per well. Representative experiment showing n = 3 wells from each condition from a single enteroid line. ****p < 0.0001. Bars show mean ± SEM; two-way ANOVA with Tukey correction for multiple comparisons (b); one-way ANOVA with Tukey correction for multiple comparisons (d, e). Each experiment repeated with at least three different enteroid lines. Specific conditions were excluded from statistical analysis if the data from one or more samples was labeled as not detectable. Source data are provided as a Source Data file.