Figure 6.

Gln enhances ISC WNT signaling in vivo and in vitro

(A and B) Crypt domains and surface areas quantification of primary enteroids from the mice in the weaned group and the Ala-Gln group cultured with indicated concentrations of R-spondin (n = 18, 18 primary enteroids from 3 independent mice per condition were analyzed, means ± SD; ^∗p < 0.05, ^∗∗p < 0.01; one-way ANOVA, results of 3 independent experiments).

(C) Representative images of primary enteroids at 96 h in vitro culture are shown. Scale bars, 100 μm.

(D) Representative image of crypts isolated from the jejunum of the early weaning mice with or without Ala-Gln supplementation. Scale bars, 10 μm.

(E) Representative image of jejunum crypts immunofluorescence stained with β-catenin (green), OLFM4 (red), and DAPI (blue). Scale bars, 40 μm.

(F) Western blot analysis of β-catenin protein expression in jejunum crypts (n = 3, separated crypts from 3 independent mice per group, results of 3 independent experiments).

(G) Representative bright-field images of enteroids treated with/without Gln and IWP-2. Scale bars, 200 μm.

(H and I) Crypt domains and surface area of enteroids were measured at 72 h (n = 12, 12 enteroids per condition were analyzed, means ± SD; ^∗p < 0.05, ^∗∗p < 0.01; one-way ANOVA, results of 3 independent experiments).

(J) Representative images of primary enteroids immunofluorescence stained with β-catenin (green) and DAPI (blue) at 96 h. Scale bars, 100 μm.

(K) qRT-PCR analyses on the expression of multiple WNT-target genes in the enteroid crypt domains at 72 h (n = 8, 8 wells of enteroids per group, means ± SD; ^∗p < 0.05, ^∗∗p < 0.01; one-way ANOVA, results of 3 independent experiments).

(L) Western blot analysis of cytoplasm and nucleus β-catenin levels of the enteroid crypt domains at 96 h (n = 3, 3 wells of enteroids per group, results of 3 independent experiments).