1. Volume 133, Issue 3, Pages 976-984 (September 2007)
RB Loss Abrogates Cell Cycle Control and Genome Integrity to Promote Liver Tumorigenesis 
Christopher N. Mayhew, Scott L. Carter, Sejal R. Fox, Charlene R. Sexton, Christopher A. Reed, Seetha V. Srinivasan, Xiangdong Liu, Kathryn Wikenheiser–Brokamp, Gregory P. Boivin, Ju–Seog Lee, Bruce J. Aronow, Snorri S. Thorgeirsson, Erik S. Knudsen 
Gastroenterology 
Volume 133, Issue 3, Pages (September 2007)
DOI: /j.gastro
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2. Figure 1
Effective RB ablation in 15-day old Rbf/f;albcre+ mice. (A) PCR analysis of genomic DNA using primers surrounding the loxP sites flanking Rb exon 19. (B) Total nuclear protein was extracted and used for immunoprecipitation/immunoblot (upper panel) or immunoblot (bottom panel) analysis of RB protein levels.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Enhanced susceptibility to liver tumorigenesis in Rbf/f;albcre+ mice exposed to DEN. Rbf/f and Rbf/f; albcre+ mice were killed 6 months post-DEN. (A) Representative images of livers of male Rbf/f and Rbf/f;albcre+ mice exposed to DEN are shown (top panel). Surface tumors were scored, and the number of lesions per liver is shown (bottom panel; mean ± SEM, n = 3 to 9). No liver lesions were detected in mice not exposed to DEN. (B) Appearance of atypical foci (left panel) and adenomas (right panel) arising in a male Rbf/f;albcre+ mouse exposed to DEN. T, tumor, N, normal tissue. (C) Adenomas arising in livers of Rbf/f and Rbf/f;albcre+ mice exposed to DEN were scored, and the number of lesions per square centimeter is shown (mean ± SEM; n = 4 to 6). (D) Representative images showing BrdU incorporation in adenomas arising in both Rbf/f and Rbf/f;albcre+ mice exposed to DEN. Top panel, original magnification, ×100; bottom panel, original magnification, ×400. Arrows indicate BrdU-positive cells in tumors (T). (E) Nuclei were isolated from tumor-free liver tissue, ploidy distribution (DNA content) was analyzed by flow cytometry, and the ratio of 4N/2N for each animal was calculated. Shown is the percentage change in this ratio for Rbf/f and Rbf/f;albcre+ mice exposed to DEN vs respective Rbf/f and Rbf/f;albcre+ controls not exposed to DEN. Mean ± SEM, n = 5 or 6 mice per group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Tumors arising in Rbf/f;albcre+ livers exhibit elevated RB/E2F target gene expression. Rbf/f and Rbf/f;albcre+ mice were killed 9 months post-DEN. (A) Representative images of livers from male (top) and female (bottom) Rbf/f and Rbf/f;albcre+ mice exposed to DEN (left panel). Macroscopically visible surface tumors were scored, and the number of lesions per liver is shown (right panel; Mean ± SEM; n = 8 to 12). (B) Total RNA was extracted from discrete tumors and subjected to microarray analysis. The expression of 35 RB loss signature genes that exhibit significant up-regulation (P = .05; Student t test) in Rbf/f;albcre+ tumors is shown. (C) Total protein extracts were prepared from discrete tumors, and levels of the indicated proteins were assessed by immunoblotting.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Decreased genome integrity in liver tumors arising in Rbf/f;albcre+ mice exposed to DEN. Rbf/f and Rbf/f;albcre+ mice were killed 9 months post-DEN. (A) Tumor BrdU incorporation was detected by immunohistochemistry (mean ± SEM; n = 20 [Rbf/f] and 13 [Rbf/f;albcre+]). (B) A gene expression signature for RB loss is significantly enriched for genes associated with chromosomal instability. Cell cycle and CIN scores for RB loss signature genes were defined as previously described.23,24 (C) Nuclei were isolated from tumors arising in male Rbf/f and Rbf/f;albcre+ mice, and ploidy distribution was analyzed by flow cytometry. For each tumor, the percentage of nuclei with diploid or tetraploid genomes was calculated. The percentage of tumors containing cells harboring predominantly diploid (white) or tetraploid/aberrant (red) genomes is indicated. N = 16 (Rbf/f +DEN) and 29 (Rbf/f;albcre+ + DEN); *P < .05, 1-way ANOVA.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Elevated expression of RB loss signature genes predicts poor survival of HCC patients. (A) Relative expression of 137 RB loss signature genes in 139 human HCC samples. HCC tissues were ordered according to average expression level of the RB loss signature. HCC subclass indicates individuals expressing previously defined HCC subclasses A (red), B (blue), and HB (green). (B) Kaplan-Meier plot indicating the overall survival of 113 of the 139 HCC patients for which survival data were available. The total expression of RB loss signature genes for each patient was calculated, and patients were separated into 2 groups based on total expression levels that were either above (red line) or below (green line) the mean total expression of RB loss signature genes across all samples.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions