Figure 2.
ERAdP deficiency causes loss of DCVs in Paneth cells. (A) Upper: HE staining of duodenum, jejunum, and ileum from littermate Cnep1r1fl/fl (WT) and Vil-Cre;Cnep1r1fl/fl (Cnep1r1ΔIEC) mice. Scale bar: 20 μm. Lower: Proportion of normal and abnormal Paneth cells were quantified on the basis of whether Paneth cells displayed a typical staining pattern with distinguishable granules (normal) or disordered, depleted, and/or diffuse staining (abnormal), and numbers of Paneth cells were quantified. n = 6 mice for each group, with 20 selecting maximal crypt sections that displayed all Paneth cells within each crypt were analyzed per mouse. (B) AB-PAS staining of intestines from WT and Cnep1r1ΔIEC mice. Scale bar: 25 μm. (C) Left: 3D immunofluorescence staining of ileum crypts from littermate WT and Cnep1r1ΔIEC mice. Green: UEA-1, red: EpCAM, and yellow: DCVs’ surface fitted by Imaris 9 software. Scale bar: 35 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group). (D) Left: Electron micrograph of WT and Cnep1r1ΔIEC mice. Paneth cells were drawn with dashed white lines, normal DCVs were indicated by yellow asterisk, and abnormal DCVs were indicated by red asterisk. Scale bar: 5 μm. Right: Numbers of DCVs of WT and Cnep1r1ΔIEC mice were calculated (n = 10 mice for each group). (E–G) Immunofluorescence staining of SIs from littermate WT and Cnep1r1ΔIEC mice. Green: MUC2 (E), OLFM4 (F), and Ki67 (G); red: EpCAM; blue: DAPI. Scale bar: 20 μm. (H) Cleaved caspase-3 immunohistochemistry of intestines from WT and Cnep1r1ΔIEC mice. Apoptotic epithelial cells were indicated by black asterisk. Scale bar: 50 μm. (I) Upper left: Schematic diagram of stimulation of CCh to organoids and DIC images of organoids from littermate WT and Cnep1r1ΔIEC mice. Upper right: Area of DCVs were quantified (n = 7 mice for each group). Organoids were stimulated by CCh for 10 min and then washed. Images were captured at 0, 2, 6, 10, and 24 h. Areas of DCVs were drawn with dashed red lines, and the corresponding Paneth cells were drawn with dashed black lines. Scale bar: 20 μm. (J) GO pathways enrichment analysis of downregulated genes in Cnep1r1ΔIEC mice versus littermate WT mice. Gene ratio (%) indicates proportion of genes annotated to each GO term among the total set of significantly downregulated genes in Cnep1r1ΔIEC versus WT mouse intestinal crypts. Data in A, C, D, and I are shown as means ± SEM, and significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments.

ERAdP deficiency causes loss of DCVs in Paneth cells. (A) Upper: HE staining of duodenum, jejunum, and ileum from littermate Cnep1r1fl/fl (WT) and Vil-Cre;Cnep1r1fl/fl (Cnep1r1ΔIEC) mice. Scale bar: 20 μm. Lower: Proportion of normal and abnormal Paneth cells were quantified on the basis of whether Paneth cells displayed a typical staining pattern with distinguishable granules (normal) or disordered, depleted, and/or diffuse staining (abnormal), and numbers of Paneth cells were quantified. n = 6 mice for each group, with 20 selecting maximal crypt sections that displayed all Paneth cells within each crypt were analyzed per mouse. (B) AB-PAS staining of intestines from WT and Cnep1r1ΔIEC mice. Scale bar: 25 μm. (C) Left: 3D immunofluorescence staining of ileum crypts from littermate WT and Cnep1r1ΔIEC mice. Green: UEA-1, red: EpCAM, and yellow: DCVs’ surface fitted by Imaris 9 software. Scale bar: 35 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group). (D) Left: Electron micrograph of WT and Cnep1r1ΔIEC mice. Paneth cells were drawn with dashed white lines, normal DCVs were indicated by yellow asterisk, and abnormal DCVs were indicated by red asterisk. Scale bar: 5 μm. Right: Numbers of DCVs of WT and Cnep1r1ΔIEC mice were calculated (n = 10 mice for each group). (E–G) Immunofluorescence staining of SIs from littermate WT and Cnep1r1ΔIEC mice. Green: MUC2 (E), OLFM4 (F), and Ki67 (G); red: EpCAM; blue: DAPI. Scale bar: 20 μm. (H) Cleaved caspase-3 immunohistochemistry of intestines from WT and Cnep1r1ΔIEC mice. Apoptotic epithelial cells were indicated by black asterisk. Scale bar: 50 μm. (I) Upper left: Schematic diagram of stimulation of CCh to organoids and DIC images of organoids from littermate WT and Cnep1r1ΔIEC mice. Upper right: Area of DCVs were quantified (n = 7 mice for each group). Organoids were stimulated by CCh for 10 min and then washed. Images were captured at 0, 2, 6, 10, and 24 h. Areas of DCVs were drawn with dashed red lines, and the corresponding Paneth cells were drawn with dashed black lines. Scale bar: 20 μm. (J) GO pathways enrichment analysis of downregulated genes in Cnep1r1ΔIEC mice versus littermate WT mice. Gene ratio (%) indicates proportion of genes annotated to each GO term among the total set of significantly downregulated genes in Cnep1r1ΔIEC versus WT mouse intestinal crypts. Data in A, C, D, and I are shown as means ± SEM, and significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments.

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