Supplementary Figure legend
Supplementary Figure 1. Expressions of GRAIL mRNA and protein in the peripheral blood CD4+ T cells of Il-10-/- mice. A. The levels of grail mRNA in CD4+ T cells isolated from the peripheral blood of Il-10+/- and Il-10-/- mice were evaluated by qRT-PCR. n=6, each. *, P < 0.05. B. The levels of GRAIL protein in the peripheral blood CD4+ T cells of Il-10+/- and Il-10-/- mice were analyzed by western blot. Representative blots of four independent experiments were shown. Relative protein levels were determined by densitometry as indicated below the blots.
Supplementary Figure 2. Evaluation of GRAIL protein and mRNA in the intestine of the patients with CD. A. The paraffin-embedded sections of inflamed colon specimens of the patients with CD and unaffected colonic mucosa of colon cancer patients (control) were evaluated by immunohistochemistry using anti-CD4 and anti-GRAIL antibodies. The percentage of GRAIL-positive in CD4-positive cells was analyzed in 4 high power fields and the ratio was significantly higher in CD than control subjects (*; P 0.01). B. Tissue samples of the inflamed colon of the patients with CD and unaffected colonic mucosa of colon cancer patients were obtained and the grail mRNA expression was determined by qRT-PCR. The grail mRNA expression was not significantly different between control and CD.
Supplementary Figure 3. Evaluation of ubiquitin-related molecules in CD4+ T cells of LP and other lymphoid organs. A. The levels of otub-1 mRNA in the CD4+ T cells of Il-10+/- and Il-10-/- mice were analyzed by qRT-PCR. Data were the mean + SEM from 4 independent experiments. B. Ubiquitination of GRAIL was determined in the CD4+ T cells from the SP and LP of Il-10+/- or Il-10-/- mice by immunoprecipitation with an anti-GRAIL antibody followed by immunoblotting with an anti-ubiquitin (Ub) antibody. Ubn; ubiquitinated fragment.
Supplementary Figure 4. Regulatory effect for proliferation of T cells by GRAIL-overexpressing cells. Either GRAIL-GFP expressing or control (GFP) DO11.10 cells were sorted by flow cytometry. Murine CD4+ splenocytes (2×105 cells) fluorescent-labeled with CellTrace™ Violet Cell Proliferation kit (Life technologies, Carlesbad, CA, USA) were co-cultured in vitro with GRAIL-expressing or control DO11.10 cells (2×105 cells) in 24 well culture plates in the presence of anti-CD3 mAb (2 μg/ml) and recombinant mouse IL-2 (20 U/ml) for 48 h. Fluorescence was analyzed by FACSCantoTMII (BD Biosciences) and discrete peaks in the histogram represent successive generations of cells. Shaded area shows violet fluorescence of the cells cultured with control cells and the solid line shows that with GRAIL-expressing cells. Shift of histogram toward right indicates decreased proliferation. A representative figure of 6 independent experiments is shown.
Supplementary Figure 5. The cellular profile of mice injected with GRAIL-overexpressing cells by flow cytometry. Mononuclear cells of SP and LP were stained with pacific blue-labeled anti-CD4, PE-Cy7-labeled anti-CD11b, phycoerythrin-labeled B220 and FITC-labeled anti-CD8 antibodies and the fluorescence was determined by flow cytometry FACSCantoTMII (BD Biosciences). The proportion was not significantly different between mice injected with GRAIL-overexpressing and control cells. Representative figures of 3 independent experiments are shown.
Supplementary Figure 6. Myeloperoxidase assay and in vivo imaging of mice transfected with GRAIL-overexpressing and control cells. A. Cellular activity determined by myeloperoxidase (MPO) assay. MPO activity of the colon of mice transferred with GRAIL-overexpressing (pAcGFP-GRAIL) and control cells (GFP, 7 each) was investigated using Myeloperoxidase Chlorimetric Assay Kit. The MPO activity was not significantly different between the two groups. B. Localization of fluorescent-labeled cells determined in vivo imaging system (IVIS®: Perkin Elmer, Hopkinton, MA, USA) in vivo. Accumulation of fluorescence (shown as red) was mainly detected in the abdomen of mice with GRAIL-overexpressing cells.