Figure 5
Loss of Scribble in IECs is associated with the accumulation of ROS and depletion of components of the autophagy signaling pathway. Colitis was induced by providing mice with 7 days of free access to drinking water containing 2.5% DSS; animals then were euthanized, colons were recovered, and colonic crypt cells were isolated. (A) Histograms (left) and MFI quantification (right) of ROS. (B) Left: Representative images of colonic organoids derived from tissues of DSS-treated mice were cultured for 5 days in the absence and presence of the antioxidant N-acetyl-l-cysteine (NAC). Right: The diameter of the resulting organoids were measured using ImageJ. (C) Representative images of vertical sections of colon samples follwing immunofluorescence (IF) staining for LC3 (green; left column) alone or merged with staining for DNA (DAPI; right column). (D) Relative mRNA levels of Scribble and autophagy-related genes in colonic crypt cells, as assessed by qRT-PCR. Values were normalized to those of a housekeeping gene. (E) The levels of Scribble and autophagy-related proteins was assessed by western blotting. Left: Representative blot strips following hybridization with antibodies against the respective proteins. Hsp90 was used as a loading control. Right: Relative expression as determined using ImageJ. Values were normalized to thatof the loading control (Hsp90) in the respective sample. (F) Colonic organoids were incubated (for 10 h on Day 3) in the absence (Control) or presence (Rapa) of 100 μM rapamycin. The organoids then were exposed to the MitoSOX dye probe. Left: Representative images of red fluorescence, photographed using an inverted microscope. Right: The fluorescence intensities of the organoids were measured and analyzed using ImageJ. 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, n.s. no significant.
