Liver fibrotic area was not observed in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Liver fibrosis was determined by Sirius red staining (red) in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice at 4 (upper), 12 (middle), and 20 (lower) weeks in the livers of <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA or CDAA diet. Fibrotic changes were not observed in <i>Nrd1^+/+</i> or <i>Nrd1^−/−</i> mice fed the CSAA diet (left). Fibrotic changes were prominent in <i>Nrd1^+/+</i> mice, but not in <i>Nrd1^−/−</i> mice fed the CDAA diet (right). Bars indicate 100 µm. B. Quantification of fibrotic areas. Fibrotic areas was observed and increased in a time-dependent manner only in the livers of <i>Nrd1^+/+</i> mice fed the CDAA diet. n = 5–8, each. *<i>P</i><0.05.</p

TNF-α was not sufficiently secreted from in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. A liver fragment was divided into two pieces. One was directly subjected to protein extraction directly and measurement of TNF-α (produced TNF-α). The other piece was cultured in serum-free medium for 12 hours, and the supernatant was subjected to measurement of TNF-α (secreted TNF-α). The relative optical density (O.D.) to that of <i>Nrd1^+/+</i> fed the CSAA diet was determined by ELISA for TNF-α. Production of TNF-α from liver specimens were not significantly different between <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CDAA diet for 20 weeks (“produced TNF-α”). In contrast, TNF-α secreted from liver specimens was significantly increased in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks compared with those fed the CSAA diet; however, the elevation was not observed in <i>Nrd1^−/−</i> mice under the same condition (“secreted TNF-α”). *<i>P</i><0.05. B. Production of IL6 and IL1-β proteins were significantly increased only in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks compared with those in <i>Nrd1^−/−</i> mice fed the CSAA diet for 20 weeks. *<i>P</i><0.05. C. mRNA (upper, ‘produced’) and protein (lower, ‘secreted’) production of IL6 and IL1-β were significantly increased after LPS treatment in <i>Nrd1^+/+</i> mouse peritoneal macrophages. However, administration of anti-TNF-α neutralizing antibodies significantly suppressed the production of IL6 and IL1-β in the presence of LPS. *<i>P</i><0.05.</p

TNF-α was expressed in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Immunohistochemistry showed that TNF-α protein (red, arrowheads) was expressed in F4/80-positive Kupffer cells or macrophages (green, arrowheads) in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mouse fed the CDAA diet for 20 weeks (right), but not in mice fed the CSAA diet for 20 weeks (left). A blue color indicates DAPI-positive nuclei. Bars indicate 50 µm. B. The number of F4/80-positive cells/×100 high-power field (HPF) in livers slightly increased (approximately 1.2 times) only in <i>Nrd1^+/+</i> mice fed the CDAA diet (right). C. Relative expression of mRNA are shown as relative values compared to those at 0 w. The mRNA expression level of CCR2 was increased in <i>Nrd1^+/+</i> mice fed the CDAA diet, and the levels were significantly higher than respective values in <i>Nrd1^−/−</i> mice fed the CDAA diet. *<i>P</i><0.05.</p

Liver fibrogenesis was not observed in <i>Nrd1^−/−</i> mice fed the HFD.

<p>A. Steatosis was observed in both <i>Nrd1^+/+</i> and <i>Nrd1^–/–</i> mice after 20-week HFD administration (right), but not in those fed a normal control diet (left). Bars indicate 100 µm. B. Quantification of triglyceride in the liver. Triglyceride was elevated in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^–/–</i> mice after 20-week HFD administration, although it was significantly higher in <i>Nrd1^+/+</i> mice. n = 4, each. *<i>P</i><0.05. C. Serum ALT levels were significantly elevated in <i>Nrd1^+/+</i> mice upon administration of the HFD, but were not elevated in other mouse groups. *<i>P</i><0.05. D. Fibrotic area was less prominent in <i>Nrd1^−/−</i> mice than in <i>Nrd1^+/+</i> mice (right). Bars indicate 100 µm. E. Fibrotic area was observed only in the livers of <i>Nrd1^+/+</i> mice fed the HFD (right). n = 5, each. *<i>P</i><0.05.</p

CDAA diet did not cause steatohepatitis in <i>Nrd1^−/−</i> mice.

<p>A. Serum ALT was not elevated in both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA diet (left). Serum ALT levels were significantly elevated in <i>Nrd1^+/+</i> mice, but were not elevated in <i>Nrd1^−/−</i> mice upon administration of the CDAA diet (right). Each value depicts the mean ± standard errors (n = 5–15, each). *<i>P</i><0.05. B. Relative expression of mRNA are shown as relative values compared to those at 0 w. mRNA of TNF-α was increased in both <i>Nrd1^+/+</i> (significantly) and <i>Nrd1^−/−</i> mice fed the CDAA diet compared with those fed the CSAA diet, with no significant difference between the two groups. In contrast, the mRNA expression levels of IL6 and IL1-β were increased only in <i>Nrd1^+/+</i> mice, and these levels were significantly higher than respective values in <i>Nrd1^−/−</i> mice fed the CDAA diet. (n = 5–16, each) *<i>P</i><0.05.</p

CDAA diet caused hepatic steatosis in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice.

<p>A. Histology of the livers of <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA (left) or CDAA (right) diets. Representative changes of the liver with regard to fat deposition at 4 (upper), 12 (middle), and 20 (lower) weeks during the experiments are depicted. Fat deposits were confirmed by oil red O staining shows (orange, inset). CV indicates central vein, and PT marks portal triad. Bars indicate 100 µm. B. Quantification of triglyceride in the liver. Triglyceride in the liver was increased in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice during administration of the CDAA or CSAA diets, although it was significantly more prominent in <i>Nrd1^+/+</i> mice. n = 4–5, each. *<i>P</i><0.05. C. There was no significant difference in the liver/body weight ratio between <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice during the experiments.</p

Activated myofibroblasts were not observed in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Immunostainings for αSMA demonstrated that activated myofibroblasts were detected in <i>Nrd1^+/+</i> mice fed a CDAA diet for 20 weeks, but not in <i>Nrd1^+/+</i> mice. Activated myofibroblasts were hardly detected by administration of the CSAA diet in <i>Nrd1^+/+</i> or <i>Nrd1^−/−</i> mice. Bars indicate 100 µm. B. The number of αSMA-positive cells/×400 high-power field (HPF) in livers increased only in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks. *<i>P</i><0.05.</p

Inflammatory and fibrogenic factors were not increased in <i>Nrd1^−/−</i> mice fed the HFD.

<p>A. mRNA of TNF-α was slightly increased in both <i>Nrd1^+/+</i> (significantly) and <i>Nrd1^−/−</i> mice. In contrast to <i>Nrd1^+/+</i> mice, the mRNA expression level of IL1-β was not increased in <i>Nrd1^−/−</i> mice. *<i>P</i><0.05. B. mRNA expression levels of collagen I, collagen IV, TIMP, TGF-β, and αSMA in the livers of <i>Nrd1^+/+</i> mice fed a HFD for 20 weeks were significantly higher than the respective values of <i>Nrd1^+/+</i> mice fed the control diet. However, they were not altered by HFD in <i>Nrd1^−/−</i> mice. *<i>P</i><0.05.</p

Fibrogenic factors were not elevated in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>During CDAA diet administration, mRNA expression levels of collagen I, collagen IV, TIMP, TGF-β, and αSMA were significantly increased in the livers of <i>Nrd1^+/+</i> mice but not in those of <i>Nrd1^−/−</i> mice. Those factors were not altered by administration of the CSAA diet in <i>Nrd1^+/+</i> or <i>Nrd1^−/−</i> mice. *<i>P</i><0.05.</p

EPRAP in bone marrow–derived cells attenuates colitis and colitis-induced tumorigenesis.

<p>Four kinds of chimeric mice were generated by adoptive bone marrow transplantation: <b>(A)</b> Representative photographs of colons with adoptive bone marrow transplantation. <b>(B)</b> Percent changes in colon length expressed relative to DSS-free water controls in each group. (n = 4–8 each). <b>(C)</b> Histological colitis scores (n = 4–8 each). <b>(D)</b> Representative H&E staining of rectal sections. Scale bars: 100 μm. <b>(E)</b> Representative photographs of colons at the end of AOM/DSS treatment. <b>(F)</b> The number of colonic tumors per mouse, with the size distribution (left) and the total number (right) in each group (n = 4–9 each). <b>(G)</b> EPRAP did not directly affect cell proliferation. DLD-1 cell proliferation was determined by MTS assay (over expression of EPRAP, left; knockdown of EPRAP, right). Data represent fold induction of absorbance compared with mock or negative control. All values represent means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p