Antibodies used are described in the Supporting Information. Densitometric quantification of immunoblots was performed using ImageJ 1.43 software. Because inactivation of the TGF-β signaling pathway and mutation of TP53 are JAK inhibitor common molecular events observed in human HCC, we assessed whether deletion of Tgfbr2 and Trp53 cooperate in the mouse liver to affect tumor formation. To this end, we crossed Alb-Cre transgenic mice with mice conditionally null for either Tgfbr2 and/or Trp53 to generate mice with liver-specific deletion of these genes.21-23 No liver tumors were observed in the control mice lacking Alb-Cre (Control) (Table 1). Likewise, deletion

of Tgfbr2 alone (Tgfbr2KO) did not induce liver tumors by 15 months of age. The Tgfbr2KO mice had a normal liver to body weight ratio of 0.050, which is not statistically different from the Control mice (Table 1). In contrast, deletion of Trp53, in the context of intact Tgfbr2 (Trp53KO) resulted in a significant number of mice developing tumors (P = 0.0034) as compared with the Control mice. The median lifespan for the entire Trp53KO cohort was 46.6 weeks, whereas the median lifespan for the subset of mice with tumors was 22.7 weeks. Survival curves illustrate that 52% of Trp53KO mice died by 50 weeks of age (Fig. 1). Additionally, the liver ZVADFMK to body weight ratio was increased

nearly 2× (P = 0.0002) in the Trp53KO cohort, presumably secondary to the tumor load present in the Trp53KO mice (Table 1). Histological analysis of the primary tumors from the Trp53KO livers revealed the tumors to be both HCC and cholangiocarcinoma (CC) (Fig. 2). The Montelukast Sodium tumors consisted of a variety of histologic subtypes, ranging from trabecular HCC with necrosis to CC with necrosis and fibrosis. Biliary hyperplasia, cholangiohepatitis, multifocal coagulative necrosis, oval cell hyperplasia, and arterial thrombosis were also noted in the adjacent

liver tissue. Of the 12 liver tumor-bearing mice, two also had multiple lung metastases that likely arose from large primary CCs. In light of the known common occurrence of TGF-β signaling inactivation and TP53 mutation in human HCC and the development of HCCs and CCs in the Trp53KO mice, we assessed the effect of Tgfbr2 deletion on liver tumor formation in these mice. Livers from mice with both inactive p53 and Tgfbr2(Trp53KO;Tgfbr2KO) were analyzed. Interestingly, the double knockout mice displayed a survival curve similar to the Control and Tgfbr2KO mice (Fig. 1). Additionally, fewer mice developed liver tumors by 15 months (Table 1, P = 0.0265) compared with the Trp53KO mice. The median lifespan for the mice with tumors was 71.6 weeks. Furthermore, the liver to body weight ratio of the tumor-bearing Trp53KO;Tgfbr2KO mice was significantly lower than the ratio of tumor-bearing Trp53KO mice (P = 0.0149).