Figure 3
Loss of Scribble in IECs impairs epithelial barrier integrity in DSS-induced colitis. Scribf/f and ScribΔIEC mice (8 weeks old) were provided with free access to drinking water containing 2.5% DSS; after 7 days, the animals were euthanized. (A) Representative images of fluorescence from FITC-dextran accumulation in the surface of the colon mucosa and graph of data for the level of FITC-dextran in serum. (B) Immunofluorescence (IF) staining for the epithelial marker E-cadherin (green) in the surface of the colon mucosa, both alone and merged with DAPI staining for DNA (blue). (C) Left: Expression of tight junction proteins (E-cadherin and occludin-1) and Scribble in colon tissues, as assessed by western blot using antibodies against the respective proteins. The levels of Hsp90, a housekeeping protein, were measured as a loading control. Right: Graph of data for relative protein expression, as obtained by measuring (using ImageJ) the band intensity in the western blot; values were normalized to those for the Hsp90 loading control in the respective lane. (D) Representative transmission electron microscope (TEM) images of tight junctions in colon epithelial cells. Images on the right are enlarged from the boxes indicated in the images on the left. (E) TUNEL assay for colon tissue of mice. Left: Representative images of TUNEL staining (green) in the surface of the colon mucosa, both alone and merged with DAPI staining for DNA (blue). Right: Graph of data for TUNEL staining. (F) The expression of caspase-3, both full-length and cleaved (C-caspase-3) in colonic crypt cells, as assessed by western blot using antibodies against the respective proteins. The levels of Hsp90, a housekeeping protein, were measured as a loading control. Left: Immunoblot. Center and right: Graphs of data for protein expression, as obtained by measuring (using ImageJ) the band intensity in the western blot. The center graph shows values normalized to those for the Hsp90 loading control; the right graph shows ratio of cleaved:intact caspase-3. Data are presented as mean ± SEM; comparisons were conducted by two-tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significant.
