Figure 1.
Aberrant crypt foci formation in AOM-treated wild type allelic (Sep15+/+), heterozygous (Sep15+/−) and Sep15 knockout (Sep15−/−) mice.
(A) Number of ACF per colon in mice fed 0.1 µg selenium/g diet (mean±SEM; N = 12 per genotype); (B) number of aberrant crypts per focus in mice fed 0.1 µg selenium/g diet (mean±SEM; N = 12 per genotype); (C) numbers of ACF in Sep15 knockout mice on 0, 0.1 or 2.0 µg selenium/g diet (mean±SEM, N = 8–10).
Figure 2.
Analysis of selenoprotein mRNA expression in colonic epithelia of wild type allelic (Sep15+/+) and Sep15 knockout (Sep15−/−) mice.
Mice were maintained on 0, 0.1 or 2.0 µg selenium/g diet and mRNA levels were measured using real-time RT-PCR. Values are means±SEM; N = 4 per genotype per diet. Letters indicate statistically significant differences (two-way ANOVA).
Figure 3.
Protein expression of selenoproteins in colonic epithelia.
Wild type allelic (Sep15+/+), heterozygous (Sep15+/−) and Sep15 knockout (Sep15−/−) mice were maintained on an 0.1 µg selenium/g diet. (A) Sep15 (80 µg protein/lane); (B) the Sep15 homolog SelM (40 µg protein/lane); and (C) TR1 (40 µg protein/lane); (D) GPx1 (40 µg protein/lane). Sep15+/+ and Sep15−/− mice maintained on diets with 0, 0.1 or 2.0 µg selenium/g diet: (E) Sep15+/+ mice, Sep15 & GPx2 (75 µg protein/lane); (F) Sep15−/− mice, SelM (75 µg protein/lane); (G) Sep15−/− mice, GPx2 (75 µg protein/lane). β-actin was used as a loading control. All experiments were carried out in triplicate.
Figure 4.
Ingenuity network analysis of genes regulated by knockout of Sep15 in mice and validation of GBP-1 mRNA expression in colonic epithelia of Sep15 knockout mice.
(A) “Cellular development, growth and proliferation” was identified as the top affected network. Genes depicted in red have up-regulated expression and genes in green have down-regulated expression in Sep15 knockout animals compared to wild type littermate controls. Molecules that are not user specified, but are incorporated into the network through relationships with other molecules, are shown in white. Relative GBP-1 mRNA expression in (B) mouse colonic epithelia of Sep15−/− and Sep15+/+ littermate control mice fed 0, 0.1 or 2.0 µg selenium/g diet (N = 8/genotype); and (C) in murine colon cancer CT-26 cells as determined by quantitative RT-PCR analysis. Means not sharing a common letter are statistically significant (p<0.0001).
Figure 5.
GBP-1 expression in mouse colonic epithelia.
GBP-1 expression was examined by Western blots in colonic epithelia of (A) Sep15 knockout (Sep15−/−), heterozygous (Sep15+/−) and wild type allelic (Sep15+/+) littermate control mice on 0.1 µg selenium/g diet, (B) Sep15−/− mice on 0, 0.1 or 2.0 µg selenium/g diet, and (C) Sep15+/+ litter mate controls on 0, 0.1 or 2.0 µg selenium/g diet. Protein extracts were loaded at 40 (A, B) or 60 µg (C) per lane. β-actin was used as the loading control. All experiments were carried out in triplicate.
Figure 6.
(A) Interferon-γ levels in serum of Sep15 knockout mice (Sep15−/−) compared to heterozygous (Sep15+/−) and wild type (Sep15+/+) litter mate controls (N = 5 per genotype) on 0.1 µg selenium/g diet as measured by ELISA. (B) Interferon-γ levels in colonic epithelia of Sep15−/− mice compared to Sep15+/− and Sep15+/+ litter mate controls (N = 5 per genotype) on 0.1 µg selenium/g diet as measured by quantitative real-time PCR. (C) Interferon- γ mRNA levels in murine colon cancer CT26 cells with targeted down-regulation of Sep15 (shSep15) compared to plasmid-transfected control cells as measured by real time RT-PCR.
Figure 7.
β-catenin protein expression in colonic epithelia of wild type (Sep15+/+) (N = 4), heterozygous (Sep15+/−) (N = 3) and Sep15 knockout (Sep15−/−) mice (N = 4) on 0.1 µg selenium/g diet.
Cell lysates from colonic epithelia were loaded at 48 µg per lane. β-actin was used as the loading control.