Fig 2: Functional enterocyte markers at protein level in mutant mice. (A) Representative Western blot images and quantification of VILLIN, downregulated in adenoma (DRA), sodium-hydrogen exchanger 3 (NHE3), sodium-glucose cotransporter 1 (SGLT1), and glucose transporter 2 (GLUT2) with respect to β-actin (Actin) in intestinal tissue lysates from control (Con) and mutant (Mut) mice, with decreased NHE3, SGLT1, and GLUT2 expression and unchanged VILLIN and DRA expression. The same blot was used to detect DRA and NHE3 protein. (B) Representative images of immunofluorescent staining of Con and Mut intestinal tissue section showing expression of VILLIN (Red) and Draq5 staining for nuclear DNA (blue) (upper panel); expression of NHE3 (red) and Draq5 staining for nuclear DNA (blue) (middle panel). Representative bright-field images of immunohistochemical staining of Con and Mut intestinal tissue sections showing expression of SGLT1. NHE3 and SGLT1 lack uniform distribution along the brush border membrane in Mut tissue when compared with Con tissue, while there was no change in localization of VILLIN expression along the membrane (n = 3). Scale bar for all immunofluorescence images = 50 μm. Scale bar for bright-field images = 100 μm. (C) Representative images of electron microscopy in neonatal Con and Mut small intestinal tissue showing brush border microvilli. Red scale bar = 1 μm. The graphs describe the average well oriented microvilli height and number of microvilli per unit length from n = 3 samples. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 by Student’s t test for all results.

Fig 3: Unchanged colonic epithelial homeostasis in mutant mice. (A) Representative bright-field H&E staining images of colonic tissue section from control (Con) and mutant (Mut) mice (n = 4). Mild colonic cellular disorganization is noted along the brush border of the Mut tissue, although the overall architecture is preserved. Scale bar = 100 μm. (B) Equivalent mRNA expression for markers of glucose transporters sodium-glucose cotransporters 1 and 2 (SGLT1, SGLT2) and glucose transporters 1, 2, and 5 (GLUT1, GLUT2, GLUT5) in colonic tissue lysates of Mut mice when compared with Con mice (n = 6). (C) Equivalent mRNA expression for apical brush border markers VILLIN, aquaporin 7 (AQP7), downregulated in adenoma (DRA), sodium-hydrogen exchanger 3 (NHE3), and epithelial sodium channel alpha and beta (ENaCα, ENaCβ) as well as basolateral membrane markers ATPase beta 1 (ATPβ1) and ATPase alpha 1 (ATPα1), sodium bicarbonate cotransporter 1 (NBC1), and anion exchanger 2 (AE2) in colonic tissue lysates of Mut when compared with Con mice (n = 6).