1. Volume 131, Issue 1, Pages 179-193 (July 2006)
Deletion of the SOCS3 Gene in Liver Parenchymal Cells Promotes Hepatitis–Induced Hepatocarcinogenesis 
Hisanobu Ogata, Takashi Kobayashi, Takatoshi Chinen, Hiromi Takaki, Takahito Sanada, Yasumasa Minoda, Keiko Koga, Giichi Takaesu, Yoshihiko Maehara, Mitsuo Iida, Akihiko Yoshimura 
Gastroenterology 
Volume 131, Issue 1, Pages (July 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Enhanced STAT3 activation and reduced expression levels of SOCS3 in human HCC. (A) IL-6 and IFN-γ mRNA analyzed in the noncancerous liver tissues of patients with chronic hepatitis C (n = 20) and in the normal liver tissues of control subjects without liver disease (n = 17). (B) SOCS3 mRNA analyzed in the noncancerous (n) and cancerous (hcc) tissues of the liver of patients with chronic hepatitis C (n = 20) and in control subjects (n = 17). (C) Western blot analysis of phosphorylated STAT3 (py-stat3), STAT3, phosphorylated STAT1 (py-stat1), STAT1, phosphorylated ERK1/2 (p-erk1/2), ERK, and SOCS3 in human whole liver lysates. non-hcc; noncancerous region. hcc; cancerous region. The data are representative of 4 experiments. (D) Quantitative analysis of the phosphorylation of STAT3, STAT1, and ERK, and protein expression levels of SOCS3. The band density of Western blots was quantified by densitometry and plotted. The level of phosphorylated protein was normalized to that of nonphosphorylated protein. Details on the patients are shown in Table 1. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
Deletion of SOCS3 in the liver. (A) Wild-type SOCS3 locus, targeted SOCS3 locus in the SOCS3flox allele, and SOCS3 locus in the liver from SOCS3fl/fl mice. The positions of PCR primers A and B are shown. (B, C) The PCR products obtained with primers A and B from the nondeleted SOCS3flox locus are 1600 base pairs, and a band of 250 base pairs obtained with primers A and B indicates the Cre-mediated deletion of SOCS3 (SOCS3flox). (B) PCR analysis of genomic DNA from brain (1), heart (2), lung (3), liver (4), stomach (5), kidney (6), muscle (7), and fat (8). Upper panel, SOCS3fl/fl mouse; lower panel, SOCS3–cKO (AlbCre–SOCS3fl/fl) mouse. (C) PCR analysis of genomic DNA from separated liver cells, that is, parenchymal cells and nonparenchymal cells, derived from SOCS3–cKO and control SOCS3fl/fl mice. p, parenchymal cell. n, nonparenchymal cell. (D) Western blot analysis of pY-STAT3 and Bcl-XL in primary mouse hepatocytes stimulated with IL-6 derived from SOCS3–cKO (AlbCre–SOCS3fl/fl) and wild-type (SOCS3fl/fl) mice. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Hepatocarcinogenesis in SOCS3-deficient mice. (A, B) Liver tumors of SOCS3+/− and wild-type mice 14 months after DEN-induced tumorigenesis (n = 8). (C, D) Liver tumors of liver-specific SOCS3–cKO (AlbCre–SOCS3fl/fl) and control mice 6 months after DEN- and choline-deficient, L-amino acid diet–induced tumorigenesis (n = 6). The total number of tumors (□) including HCCs (■) was scored from 100 series of 3-mm sections from the livers of mice. Macroscopic observations after DEN treatment are shown in B and D. The arrows indicate tumors. Details of the tumors are shown in Table 2. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Proliferation of liver cells after the administration of DEN in SOCS3–cKO (AlbCre–SOCS3fl/fl) mice. (A–F) Wild-type and SOCS3–cKO mice were injected with DEN (40 μg/g body weight). (A) Serum ALT levels were measured at 24 hours after the administration of DEN (n = 6). (B) Serum IL-6 levels were measured by enzyme-linked immunosorbent assay (n = 6). (C) Representative immunohistochemical staining of cyclin D1 in wild-type and SOCS3–cKO mouse liver tissue sections (magnification, 200×). In the right panel, the percentage of cyclin D1–positive hepatocytes is shown (n = 4). (D) Western blot analysis of pY-STAT1, pY-STAT3, p-ERK1/2, Bcl-XL, and SOCS3 in whole liver extracts from mice at the indicated time points. The data are representative of 4 experiments. (E) Immunostaining for Bcl-XL in the liver after DEN injection. Scale bar, 100 μm. (F) Relative expression levels of SOCS1, SOCS3, Bcl-XL, Bcl-2, Bcl-10, c-Myc, cyclin D1, and VEGF mRNA analyzed by real-time RT-PCR (n = 6). The value for each group was normalized to the glyceraldehyde-3-phosphate dehydrogenase value. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
Liver injury and activation of cytokines and natural killer T cells after the administration of con A. (A–E) Wild-type (SOCS3fl/fl) and SOCS3–cKO (AlbCre-SOCS3fl/fl) mice were injected with con A (15 μg/g body weight). (A) The serum ALT levels were measured at the indicated time points (n = 6). □, SOCS3fl/fl; ■, AlbCre-SOCS3fl/fl. (B) Photomicrographs of representative mouse livers 20 hours after the injection of con A with H&E staining and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Magnification, 200×. In the right panel, the percentage of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)–positive cells is shown (n = 4). White arrows indicate massive necrosis of the liver. (C) The serum IL-6, IFN-γ, and tumor necrosis factor-α levels were measured by enzyme-linked immunosorbent assay at the indicated time points (n = 6). (D) Hepatic lymphocytes were isolated, stained with anti-CD4 and anti-CD69 monoclonal antibody (left panel) or anti-NK1.1 and anti-CD3 monoclonal antibody (right panel), and were analyzed by flow cytometry. The upper-right quadrant in each panel shows CD4+CD69+ or NK1.1+CD3+ double-positive cells (percentage of the total hepatic lymphocytes). The data are representative of 3 experiments. (E) Means ± SEM from 3 mice at each time point (n = 3). ○, SOCS3fl/fl; •, AlbCre-SOCS3fl/fl. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
STAT1 and IRF-1 activation are attenuated, but STAT3 and Bcl-XL activation are enhanced and prolonged in con A–induced hepatitis in SOCS3–cKO (AlbCre-SOCS3fl/fl) mice. (A–D) Wild-type and SOCS3–cKO mice were injected with con A (15 μg/g body weight). (A) Western blot analysis of pY-STAT1, pY-STAT3, IRF-1, Bcl-XL, p-IKBα, and cleaved caspase 3 in whole liver lysates from mice. Total liver protein extracts from con A–treated wild-type and SOCS3–cKO mice were analyzed. The data are representative of 3 experiments (n = 6). (B) Quantitative analysis of the phosphorylation of STAT3 and STAT1 expression levels. The band density of Western blots was quantified by densitometry and plotted (n = 6). □, SOCS3fl/fl; ■, AlbCre-SOCS3fl/fl (C) Relative expression levels of SOCS1, SOCS3, Bcl-XL, and IRF-1 mRNA analyzed by real-time RT-PCR. The value for each group was normalized to the glyceraldehyde-3-phosphate dehydrogenase value (n = 6). (D) Immunostaining for pYSTAT3 and Bcl-XL in the liver after con A injection. □, SOCS3fl/fl; ■, AlbCre-SOCS3fl/fl. Scale bars, 20 μm for pYSTAT3 and 100 μm for BcL-XL. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions