FIGURE 3.

Histological and messenger RNA (mRNA) analysis of livers 9 months after PBS or DEN treatment confirm the presence of HCC in Zhx2 ^wt mice and the absence of tumors in Zhx2 ^KO mice. (A,B) Hematoxylin and eosin staining (A) and immunohistochemical staining for glutamine synthetase (B) in Zhx2 ^wt and Zhx2 ^KO male mice after PBS or DEN treatment. (C–E) Quantitation of hepatic Zhx2 (C), Gpc3 (D), and G6PC (E) mRNA levels by real‐time quantitative polymerase chain reaction (PCR) in PBS‐treated or DEN‐treated Zhx2 ^KO mice (n = 8 and 10, respectively) or livers from PBS‐treated Zhx2 ^wt mice (n = 9) or tumors or nontumor tissues from DEN‐treated Zhx2 ^wt mice (n = 10). mRNA levels were normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). **p < 0.01 and ***p < 0.001. (F) Zhx2 protein levels were analyzed by western blot of liver lysate from PBS‐treated or DEN‐treated Zhx2 ^KO mice or livers from PBS‐treated Zhx2 ^wt mice or tumors or nontumor tissues from DEN‐treated Zhx2 ^wt mice. Blots were reprobed with antibodies against GAPDH. The Zhx2 protein levels were quantified using ImageQuant TL Image Analysis Software (GE Healthcare) and normalized to GAPDH. Abbreviations: KO, knockout; nt, nontumor tissues; tu, tumors; WT, wild‐type.