Indispensable role of the Ubiquitin-fold modifier 1-specific E3 ligase in maintaining intestinal homeostasis and controlling gut inflammation

a Ufl1 and Ufbp1 are highly expressed in intestinal Paneth cells. Ufl1 and Ufbp1 proteins were examined by immunohistochemistry of ileal sections. Ufl1f/f:ROSA26-CreERT2 and Ufbp1f/f: ROSA26-CreERT2 mice were treated with tamoxifen for 5 days and killed at 2 weeks after initial treatment of tamoxifen. Goblet cells are labeled with “G”, while Paneth cells are marked with “P”. b Tamoxifen-induced deletion of Ufl1 exon 7. c. Ufl1 mRNA level in Ufl1-deficient intestine. Quantitative RT-PCR was performed using total RNA isolated from small intestinal crypts and the primers specific for deleted exon 7. The p values were determined by unpaired t-tests. p？<？0.001 (n？=？6). d Ufl1 protein level in wild-type and Ufl1-deleted intestines. e H&E and Alcian/PAS staining of small intestinal sections and quantitation of Paneth and goblet cells in the crypts. At least 100 crypts were counted from 6 mice of each genotype in a double blinded manner. The average numbers of Paneth and goblet cells per crypt were scored. The p values were determined by unpaired t-tests. f H&E and Alcian/PAS staining of the colonic sections and quantitation of goblet cells in the crypts (n？=？6). Quantitation was conducted as described in “e”. g Lysozyme staining of ileal sections. h Immunoblotting of lysozyme in Ufl1-deficient crypts. Crypts were isolated accordingly and the lysates were subjected to immunoblotting of indicated antibodies. Ufmylation conjugates were indicated by asterisks. Scale bar is 20 μm. i Electron micrographs of the crypts of wild-type and Ufl1-deficient mice. Scale bar is 5？μ