Figure 1: Human TRIM31 is enriched in the mitochondria and downregulated in Crohn’s disease.

(a,b) TRIM31 is highly expressed in colonic and intestinal epithelial cell lines. The expression of TRIM31 was analysed by RT-PCR (a) or immunoblot analysis (b) in various human cell lines: SW480 (colon), HT-29 (colon), and Caco-2 (intestine), A549 (lung), HeLa (cervix), U937 (lymphocyte), MCF-7 (breast), HFF (fibroblast), JEG-3 (placenta), 293T (embryonic kidney), Caki-2 (kidney) and AGS (stomach). Graph in a, quantification of TRIM31 mRNA levels normalized against GAPDH (mean±s.d.). (c) TRIM31 is enriched in the mitochondria. Immunofluorescence assay (IFA) of HeLa cells transfected with TRIM31-Myc. Cells were immunostained with anti-Myc antibody (green) and anti-TOM20 antibody (red), followed by AlexaFluor 488- or AlexaFluor 568-conjugated antibodies. TOM20 serves as mitochondrial marker. Scale bar, 5 μm. (d) Mitochondrial localization of TRIM31-Myc. Cytosolic and mitochondrial fractions were isolated from TRIM31-Myc-expressing HeLa cells, and then subjected to immunoblot analysis using anti-Myc antibody. Anti-TOM20 and anti-Tubulin antibodies were used to designate the mitochondrial and cytoplasmic compartments, respectively. (e) TRIM31 is associated with the mitochondrial membrane. Isolated mitochondria from TRIM31-Myc-expressing HeLa cells were treated with 0.1 M NaCl or 0.1 M Na₂CO₃ (pH 11.5) for 20 min on ice. After centrifugation, the supernatant (S) and pellet (P) were analysed by immunoblotting using anti-TRIM31 and anti-TOM20 antibodies. (f) Representative immunohistochemical analysis of human TRIM31 on tripled Crohn’s disease (CD #1, #2, #3) and adjacent normal tissues from colon cancer (Con#1, #2, #3). The ileal mucosa biopsies of the distal small intestine from normal or Crohn’s disease patients were stained with anti-TRIM31 antibody. Scale bars, 100 μm. Right graph, Immunohistochemical score (IHS) of TRIM31 in normal (n=3) or Crohn’s disease patients (n=8). Detailed patient information is presented in Supplementary Table 2. IHS was equal to the quantity scores multiplied by the staining intensity scores. *P<0.01 (Student’s t-test). (g) TRIM31 mRNA levels were determined by RT-PCR on tripled Crohn’s disease (CD #1, #2, #3) and adjacent normal tissues from colon cancer (Con#1, #2, #3). (h) TRIM31 mRNA levels were normalized against GAPDH in the graph. *P<0.01 (Student’s t-test). All data are representative of at least three independent experiments (mean±s.d. in f,h).