Fig. 5

LARGE2 expression of α-DG O-glycosylation are enriched in the Wnt-driven stem/progenitor compartment of human colon epithelium. A) Human colonic organoids (PDOs) embedded in Matrigel and maintained in WREN (Wnt, R-Spondin, EGF, Noggin) medium. Scale bar represents 20 μm. B) qRT-PCR analysis of the indicated genes in PDO1 maintained in WREN (=STEM) or differentiation medium (DIFF, EN). Results are shown as mean ± SD (n = 3). *** p < 0.001; **** p < 0.0001. See Additional file 8A,B for experiments on PDO2 and PDO3. C) Immunoblot analysis of α-DG from WGA-AE purified glycoproteins of PDO1, cultivated in WREN or EN media for 72 h. WCL were used for analysis of β-DG, PTK7, and β-actin. D-G) FACS profile and Taqman™ qRT-PCR analysis from PDO single cells -stained with APC-coupled antibody for PTK7 (D,E) or from human crypt epithelial cells, stained against EPHB2 (F,G). Control staining for viable cells (DAPI) was performed to define the PTK7 or EPHB2-negative fraction (Ctrl). In D) neg: PTK7 negative, lo: PTK7 low, hi: PTK7 high. In F) hi: EPHB2 high, med: EPHB2 medium, low: EPHB2 low cell fraction. Error bars in E and G indicate mean ± SD (n = 3 technical replicates). See Additional file 8D,E for data on PDO2 and PDO3. H,I) Immunohistochemistry (IHC) analysis of α-DG (IIh6 antibody) on FFPE human colonic (H) or rectal (I) mucosa. Black arrowheads: crypt base specific staining; clear arrowheads: fading or loss of staining. Scale bars represent 100 μm (H) and 50 μm (I). J, K) In situ hybridization (ISH) with a Large2-specific probe (J) and IHC staining of glycosylated α-DG (K) on mouse small intestinal FFPE tissue (Ileum). Scale Bar represents 100 μm. Black arrowheads: staining in crypts; clear arrowheads: lack of staining in villi. See Additional File 9 for control staining (no probe) and additional data