Figure 4.

Gln deprivation disables the functions of ISCs ex vivo and in vitro

(A and B) Quantification of crypt domains (A) and surface area (B) per primary enteroids cultured in Gln-free medium supplemented with 2 mM Ala, 2 mM Gln, or 2 mM Ala-Gln were measured at 96 h (n = 12, 12 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 of immunofluorescence stained with OLFM4 (green) and DAPI (blue) at 96 h. Scale bars, 100 μm.

(D) Representative of images of primary enteroids at 48 and 96 h. Scale bars, 100 μm.

(E and F) Quantification of crypt domains (E) and surface area (F) of the stable-passaged enteroids in Gln-free medium supplemented with 2 mM Ala, 2 mM Gln, or 2 mM Ala-Gln were measured at 96 h (n = 12, 12 enteroids per condition were analyzed, means ± SD; ^∗p < 0.05, ^∗∗p < 0.01; one-way ANOVA, results of 4 independent experiments).

(G) Representative images of enteroids immunofluorescence stained with OLFM4 (green) and DAPI (blue) at 96 h. Scale bars, 100 μm.

(H) Representative bright-field images of the 50 more time-passaged enteroids in medium containing 0 mM Gln, 2 mM Gln, 2 mM Ala-Gln, or 2 mM Ala for 72 h, and the enteroids that were growing in 0 mM Gln medium were switched to 2 mM Gln supplemented medium for an additional 48 h culture. Scale bars, 100 μm.

(I) Representative images of the in vitro enteroids immunofluorescence stained with OLFM4 (green) and DAPI (blue) at 120 h. Scale bars, 100 μm.