Figure 3. VKO mice have lower number of extruding cells at villus tip.

(A) Stimulated apoptosis in response to 8Gy radiation was measured in VKO and WT littermates. Apoptotic cells were defined using TUNEL assay. A significant decrease in the number of apoptotic cells at the villus tips was noted in VKO mice compared to their WT littermates (p < 0.05, n = 6). Arrowheads indicate apoptotic cells identified by TUNEL assay in the crypts (red) and villus tips (yellow). (B) Histological examination of H and E-stained ileum from WT and VKO mice for extrusion showed extrusion in WT mice (arrowheads) but not VKO mice. VKO mice lack cell extrusion from villus tips but show apoptotic bodies along the crypt-villus axis (arrowhead). WT mice show cell extrusion from villus tips (arrowheads). (C) Transmission electron microscopy shows (a) extruding cells (E) in SI of WT littermates while (b) VKO mice show apoptotic bodies (A) and (c) dying cells with crescent nuclei (C). Data are from one representative experiment typical of eight other with similar results. (d) TUNEL assay shows apoptotic cells along the length of the villi (arrowheads) of VKO mice. Data are from one representative experiment typical of eight other with similar results. (D) Enteroids from VKO and WT littermate show the apoptotic response of the small intestinal epithelium to CPT treatment. Deletion of full-length villin (VKO) increases apoptotic cells in intestinal epithelium in response to CPT treatment (10 μM, 3.5 h) compared to WT mice. (a) Shows morphological changes in enteroids following CPT treatment; (b) shows changes in PI staining scored using fluorescence microscopy. The error bars are the measured S.E. Bar, 20 μM. (E) Western analysis of cleaved caspase-3 in WT and VKO mice untreated or treated with 3% Dextran sodium sulfate (DSS) for 5 days as described in the Methods section. Western blot with anti-tubulin antibody was performed in parallel as a quantitative control. Data are representative of three experiments with similar results.