Extended Data Fig. 2: Immune, epithelial, and stromal cell profiling upon 10 days of standard or GW3965-diet.

(a-b) Flow cytometry dot plots showing the gating strategy (a) and quantification (b) of the number of major immune cell subsets in the SI tissue of mice exposed to 10 days of standard or GW3965 diet. Cell numbers are not significantly different between diet groups in any immune cell subsets analysed. Each dot represents one mouse and data are representative of one experiment. Data are shown as bar plots with mean ± s.e.m. Unpaired two-sided t test between diet groups. The diagram was adapted from ref. ¹⁹, CC-BY 4.0. (c) Gating strategy and sorting scheme for epithelial (EpCAM⁺CD45⁻), immune (EpCAM^-CD45⁺) and stromal (i.e., DN, double negative, EpCAM⁻CD45⁻) cells processed for single cell RNA sequencing (scRNAseq). (d) UMAP distribution of scRNAseq datasets obtained from epithelial, immune and DN cells from SI of mice fed for 10 days with STD or GW3965 diet. (e-f) Left: unsupervised clustering and annotation of epithelial cells (e) and immune and stromal cells (f) in UMAP. Right: cells in the clusters shown to the left are colour-coded based on the diet treatment (e) and the combined cell compartment and diet (f). (g) Left: SI Swiss rolls shown in hematoxylin and eosin (H&E) staining and colour coded spatial transcriptomic (ST) map based on non-negative matrix factorization (NMF). Right: heatmap showing top 20 genes defining each NMF and respective annotations (i.e., NMF1: immune and intestinal epithelial cells (IEC), NMF2: muscle and NMF3: IEC). (h) Volcano plots showing the log2FC and adjusted p-values from differential expression analysis between SI scRNAseq samples from GW3965 diet- vs. STD-diet fed mice in immune, DN and epithelial cells. Genes known to be regulated by LXR are marked blue. (i) UMAP and violin plot showing expression of module scores based on genes belonging to the GO term “Inflammatory response” (GO:0006954) and their expression score in scRNAseq datasets of immune cells from STD and GW3965 samples. For scRNAseq experiment, epithelial, immune and DN cells were sorted from a pool of 3 mice. DEG were identified with Wilcoxon rank-sum test, subsampling 500 cells per group. For ST, one mouse per diet treatment was processed. MΦ, Macrophages; DC, Dendritic Cells; IEL, intra epithelial leucocytes; ILC, innate lymphoid cells; Mono, Monocytes; NΦ, Neutrophils.

Source Data