Figure 7
Dietary sphingomyelin (SM) alleviates DSS-induced colitis and is associated with increased expression (RNA and protein) of Scribble and autophagy-related genes. Colitis was induced in C57BL/6 mice by maintenance on DSS-containing water without or with sphingomyelin (SM) dietary intervention, and animals were euthanized on Day 14. Control mice were maintained on drinking water neat without dietary intervention. (A) Relative body weights (normalized to baseline). (B) DAI scores. (C) Left: Representative images of colons from experimental animals. Right: Graph of colon length data in the three groups. (D) Left: Representative images of H&E-stained colon tissue. Right: Graph of histological scores. (E) RNA was extracted from colon tissue and subjected to RNA-seq analysis. The graph shows the results of Gene Ontology (GO) annotation analysis from the comparison of DSS mice vs. DSS + SM animals. (F) RNA expression of Scribble and of the tight junction related genes CDH1 and ZO-1 was assessed by qRT-PCR. Values were normalized against the expression of a housekeeping gene. (G) Protein isolated from the colon tissue of DSS and DSS + SM mice was assessed for Scribble levels by western blotting. Left: Representative blot strips following hybridization with anti-Scribble antibody. Hsp90 was used as a loading control. Right: Blot band intensities were quantified and analyzed using ImageJ. Values were normalized to those of the loading control in the respective sample. (H) RNA was extracted from colon tissue and subjected to qRT-PCR analysis to assess transcript levels of the autophagy-related genes Atg16L, Atg7, Atg5, and Atg3 were detected by qRT-PCR. Values were normalized to those of a housekeeping transcript in the respective samples. 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.
