Fig 1: Multiomic analysis of EGF- and EREG-grown enteroids suggests altered chromatin landscape.(A) Weighted nearest neighbors (representing joint RNA and ATAC data) UMAP visualization colored by sample (fresh crypts; red, 1 ng/mL EREG [passage 0 day 10]; blue, 1 ng/mL EGF [passage 0 day 10] green; 100 ng/mL EGF [passage 0 day 10]; gray). (B) UMAP visualization of CDX2 gene expression (left) and corresponding motif activity (right). (C) Dot plot quantification of CDX2 gene expression and motif activity (MA0465.2). (D) Whole-mount and 2D immunofluorescence staining for CDX2 in fetal intestine (127-day), fetal stomach (132-day), 1 ng/mL EREG-grown enteroid (passage 1 day 10), and 100 ng/mL EGF-grown enteroid (passage 1 day 10). (E) Chromatin accessibility of intestinal genes including LGR5, DPP4, MUC2, and CHGA. (F) Chromatin accessibility of stomach genes including TFF1, TFF2, CLDN18, and S100P. (G) Schematic of ligand switching experiment. (H) RT-qPCR quantification of stomach markers (CLDN18, TFF1, and TFF2) and intestinal secretory cells (CHGA, MUC2) in enteroids grown in EGF or EREG for 2 passages, then switched to opposite ligand for 2 additional passages. Please see Methods for further details. One-way ordinary ANOVA with multiple comparisons. *: P = 0.05; **: P = 0.01; ***: P = 0.001; ****: P = 0.0001.
Fig 2: EGF-grown enteroids ectopically express gastric epithelial genes.(A) UMAP visualization of EREG-grown (1 ng/mL) enteroids (blue) and EGF-grown (100 ng/mL) enteroids (gray). (B) UMAP visualization of overall Louvain clustering and gene expression of CDX2, SOX2, PDX1, TFF1, and TFF2. Bar plot quantification of cell type abundance per Louvain cluster in each sample; colors correspond to color of cluster. (C) Dot plot quantification of gene expression shown in B, grouped by culture condition. (D) UMAP visualization of cell scoring analysis for small intestine genes (left) and stomach genes (right); see Supplemental Table 2 for gene list. (E) Violin plot quantification of cell type score as shown in D. (F) UMAP visualization of reference fetal atlas organ tissues (12) (left) and enteroid samples embedding onto reference map (right); blue dots represent 1 ng/mL EREG-grown cells, and gray dots represent 100 ng/mL EGF-grown cells. (G) Quantification of F. Bars represent the percentage of each sample that map to a specific tissue type. (H) Immunofluorescence staining for stomach gene TFF1 in human fetal intestine (127-day) and stomach (132-day), 1 ng/mL EREG-grown enteroids (passage 1 day 10), and 100 ng/mL EGF-grown enteroids (passage 1 day 10).
Supplier Page from Abcam for Anti-Estrogen Inducible Protein pS2 antibody [TFF1/2133]