Extended Data Fig. 1: Chronic ethanol alters small intestinal GCs in mice and humans.

(a–c) WT mice were fed control (n = 7–9) or ethanol-containing (n = 8) Lieber DeCarli diets for 10 weeks; 3 independent experiments. (a) EGFP-E. faecalis (5x 10⁹ CFUs) was gavaged at 3 and 0.5 h before euthanasia (n = 6); 3 independent experiments. Representative sections of EGFP (green), TMR-dextran indicating GAPs (red), and DAPI (blue) stained SI sections. Left panel: scale bar = 100 μm. Right panel (amplification of the dashed white oval on left panel): scale bar = 12 μm. Arrowheads indicate duodenal GAPs. (b) GCs were visualized using Muc2 and Periodic Acid/Schiff (PAS) staining. Percentage of duodenal Muc2-stained area in the proximal SI (PSI) and PAS-stained cells were enumerated in each villus in the PSI and each colonic crypt. (c) Representative sections of Muc2 (red; GCs) and DAPI (blue) IF images (scale bar = 250 μm), and representative PAS-stained sections showing an increased number of GCs in the PSI and colon of ethanol-fed mice (scale bar = 100 μm). (d, e) Number of positively PAS-stained cells per villus of duodenal biopsies from controls (n = 8) and patients with AUD (n = 15), and representative PAS-stained sections. Scale bar = 100 μm. (f) GSEA of GC-related genes (GALNT10, UGT1A10, PLEKHS, GALNT14, CEACAM6, MOGAT2, FUT3, USH1C, ELF3, POF1B, KRT20, FXYD3, SMIM22, FUT2, ZG16, GALNT6, PRSS8, CEACAM5, EPCAM, CDH17, TMPRSS2, C1GALT1, C1ORF210, MUC13, PKP3, B3GNT6, TMPRSS4, MST1R, C1ORF116, MUC17, SLC44A4, AP1M2, PHGR1, B3GNT3, MISP, LAD1, ST6GALNAC4, LIPH, TSPAN8, CEACAM1, AGR3, SLC26A3, ACE2, PPP1R14D, CAPN8, TPSG1, MUC5AC, MUC2) in AUD and control subjects. (g) Volcano plot of the upregulated terms in patients with AUD from the PanglaoDB Augmented 2021 gene set. Each point represents a single term from the enrichment results. P values were determined by the two-sided unpaired Student’s t-test (b, d), or Fisher’s exact test with Benjamini–Hochberg adjustment (FDR) (g). NES represents the strength; q-value was determined as the maximum Benjamini–Hochberg-adjusted p value across Fisher’s test, fGSEA, Camera, and GSVA/limma (f). Results are expressed as mean ± s.e.m. *P < 0.05, **P (or q) < 0.01. The illustrations in a, b, d, and g were created using BioRender.