Fig. 2.

Cyclin D, VEGF, cell proliferation, and neoangiogenesis are reduced in Jnk1^−/− tumors. (A) Immunohistological analysis of wild-type and Jnk1^−/− HCCs. N, noncancerous liver tissues; T, tumors. Original magnification: hematoxylin/eosin (H&E), ×50; others, ×200. (B) Frequencies of proliferating (PCNA-positive), apoptotic (TUNEL-positive), and phospho-c-Jun- or phospho-ATF2-positive cells in wild-type (Wt, n = 10) and Jnk1^−/− (n = 10) HCCs. Results are means ± SE. ∗, P < 0.05 vs. wild-type mice. (C) Expression of cell cycle proteins, p53, and VEGF. Lysates of microdissected HCCs (HCC; two separate samples) or nontumor liver tissue (Liver) from DEN-treated mice were gel-separated and immunoblotted with antibodies to the indicated proteins. (D) Effects of JNK1 on cyclin gene expression. RNA from tumors (T) and nontumor liver tissues (NT) were analyzed by real-time quantitative PCR, and the tumor (T)/nontumor tissue (NT) ratio of expression of the different cyclin genes was determined. Results are means ± SE (n = 4). ∗, P < 0.05 vs. wild-type mice (Wt). (E) Expression of VEGF in HCCs. Cryosections were immunostained with polyclonal VEGF antibody. N, noncancerous liver tissues; T, tumors. Original magnification: ×200. Distinction between tumor and noncancerous liver tissue was made by hematoxylin/eosin staining. (F) Intratumoral microvessel density in wild-type (Wt, n = 3) and Jnk1^−/− (n = 3) HCCs. Cryosections were immunostained with anti-CD31 antibody, and microvessels were counted per high-power fields (HPF; original magnification: ×400). Data are presented as means ± SE. ∗, P < 0.05 vs. wild-type mice. Corresponding representative photomicrographs (original magnification: ×200) are shown.