Figure 6

Down-regulated ASS-EGFP expression in HBx -induced hepatocellular carcinoma in Tg (ASS-EGFP, HBx) double transgenic mice. In all subfigures, mice are denoted by the ASS-EGFP designations as in Figure 1, followed by HBx to indicate double transgenics; the numerical after HBx denotes the specific mouse analyzed. The 16C line used in 16C. HBx-1 (A) and 3 F line in 3 F. HBx-1 (B) carry 3 and 2 transgene copies, respectively. (A) Localization of EGFP levels in tumor nodules observed under a fluorescence dissecting microscope. The age of the mice in months (m) when sacrificed is shown in the bottom right corner of each image. Yellow and pink arrows indicate reduced and enhanced EGFP fluorescence, respectively; the white arrow indicates apparently unaltered fluorescence levels compared with the surrounding tissue. HBx was single transgenic mouse; WT, a non-transgenic control. (B) Correlation of tumor morphology and EGFP expression. Liver frozen sections were taken from tumors of the transgenic lines. WT was a non-tumorous section from a normal mouse used as a control. Top panels, fluorescence sections; bottom panels, DAPI-stained sections. Yellow arrows indicate sites of down-regulated EGFP expression. (C) Immunohistochemical analysis of ASS and EGFP expression in tumors of the Tg (ASS-EGFP, HBx) double transgenic mice. Tumor tissues were reacted with an anti-ASS or anti-EGFP antibody and incubated with the biotinylated secondary antibody coupled with streptavidin conjugated-HRP. HRP activities were visualized by the application of chromogen DAB to produce brown coloration while the slides were counterstained with hematoxylin. (D) Histochemical analysis of selected tumor morphology of the double transgenic mice. After HE staining, the tissue samples were observed under a light microscope. Boxed field in the top panel is magnified and is shown in the bottom panel. Yellow arrows indicate central-sinusoidal trabecular pattern; red arrows indicate peripheral-sinusoidal trabecular pattern.