Figure 4

Loss of Scribble in IECs disrupts the function of crypt stem cells in DSS-induced colitis. Mice were provided with 7 days of free access to drinking water containing 2.5% DSS and then were euthanized for tissue collection. (A) IHC was performed to assess the expression of Ki67 in colon cells; the relative intensity of Ki67⁺ cells was measured using ImageJ. Left: Representative images of staining. Right: Graph of the relative intensity of staining for Ki67. (B) The levels of selected mRNAs (encoding markers of proliferation and stemness) in colon crypt cells were determined by qRT-PCR. (C) The expression of Wnt signaling-related proteins in colonic crypt cells were assessed by western blotting. Left: Representative blot strips following hybridization with antibodies against the indicated protein. Hsp90 was used as a loading control. Right: Plot of relative protiein levels as determined by western blotting; values were normalized to those of the loading control in the respective sample. (D) Left: Representative images of colon organoids derived from colonic crypts that had been isolated (on Day 7) from DSS-treated mice and subsequently cultured in vitro for 6 days. Right: The diameters of the resulting organoids were measured using ImageJ. (E) Representative images of IF staining for E-cadherin and Ki67 in colonic organoids derived from mice of the indicated genotypes. Left: staining for E-cadherin; center, staining for Ki67 (green); right, merged images. (F) The expression of c-Myc in colonic organoids was detected by western blotting. Representative blot strips are shown for membranes hybridized with antibodies against c-Myc and Hsp90 (loading control). Values between the strips correspond to relative c-Myc expression following normalization to values for the loading control in the respective sample. For all plots, 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.