Table 1.
Body and liver weights of the experimental mice.
Fig 1.
Analysis of pre-neoplastic lesions and nodules of DEN-treated mice.
Development of foci of cellular alteration (FCA), the proliferating cell nuclear antigen (PCNA)-labeling indices of the FCA, and the nodule frequency on the liver surface of DEN-treated experimental mice. (A) A representative photograph of a FCA that developed in DEN-treated WT mice at 32 weeks of age (H&E staining, left panel) and the average number of FCA in DEN-treated WT and IDO-/- mice at 20 and 32 weeks of age (right panel). (B) Representative photographs of PCNA-immunohistochemical analysis of the FCA that developed in DEN-treated WT mice and IDO-/- mice at 32 weeks of age (left panels). The PCNA-labeling indices of the FCA that developed in the experimental mice were determined by counting the number of PCNA-positive nuclei in the FCA (right panel). (C) Tumor nodules on the liver surface of DEN-treated WT and IDO-/- mice at 32 weeks. The white arrows indicate tumor nodules (left panel). The number of liver surface tumors in IDO-/- mice was significantly lower than that in wild-type mice (right panel). *, P<0.01 Data points represent the mean ± SD.
Table 2.
Incidence and multiplicity of hepatic neoplasms and FCA in the experimental mice (32 weeks-groups).
Fig 2.
Representative immunohistochemical expression (IHC) of IDO and kynurenine (KYN).
Representative images of normal tissue (top panel) of a saline treated WT mouse, of HCC (second panels) and adenoma (third panels) of a WT mouse, and of adenoma of an IDO-/- mouse (bottom panels), at 32 weeks of DEN treatment that were stained with H&E (a-d), or were immunohistochemically stained for the IDO protein (e-h) or for L-KYN (i-l). The black line in the HCC tissues demarcates HCC from non-HCC tissue. (Bar = 50 μm)
Fig 3.
Gene expression of IFN-γ, COX-2, and TNF-α in the liver.
Expression levels of (A) IFN-γ, (B) COX-2, and (C) TNF-α mRNA in hepatic tumors and in normal liver tissues of the indicated mice were measured using quantitative RT-PCR. Data points represent the mean ± SD. *, p<0.05.
Fig 4.
Expression of Foxp3 analyzed by immunohistochemistry and RT-PCR.
(A) Gene expression of Foxp3 in control and DEN-treated non-tumorous liver tissues at 32 weeks. (B) Representative pictures of the immunohistochemical analysis of FCA for Foxp3 and (C) Foxp3 mRNA expression in hepatic tumors of DEN-treated mice. mRNA expression was determined using quantitative RT-PCR. Data points represent the mean ± SD. *, p<0.05.
Fig 5.
Expression levels of CD8, FasL, perforin and granzyme B in non-tumorous liver tissues and hepatic tumors.
Expression levels of (A) CD8, (B) FasL, (C) perforin and (D) granzyme B mRNA in hepatic tumors and in normal liver tissues of the indicated mice were measured using quantitative RT-PCR. Data points represent the mean ± SD. *, p<0.05.
Fig 6.
The serum L-kynurenine/L-tryptophan ratio and tissue gene expression of IDO and TDO.
A, The serum L-kynurenine/L-tryptophan ratio (Kyn/Trp) was determined at 32 weeks by measuring the concentrations of L-kynurenine and L-tryptophan using HPLC. (B, C) Liver tissues were separated into hepatic tumors and non-tumorous tissues. Total RNA was then extracted and the expression levels of IDO and TDO mRNA were measured using quantitative RT-PCR. Data points represent the mean ± SD. * p<0.05, ** p<0.01, N.S.: not significant