fig4

Figure 4. Spheroid tumor growth suppression. (A) Dose-dependent increase of TUNEL staining. The resected tumors in mice fed with water, 2 and 4 g of BJ/kg were frozen, resected, fixed, and subjected to TUNEL experiment for confocal microscopy analysis as described in “Methods”. The apoptotic bodies (green) were counter-stained with DAPI (blue) and visualized (scale bar, 50 μm). The image in the rectangular inset of tumor sections in mice treated with 4 g of BJ/kg is zoomed in at the bottom of the panel (scale bar, 15 μm). (B) BJ increased nucleus TUNEL-positive intensities. The numbers of fluorescent TUNEL-positive cells in each field containing 100 cells as marked by DAPI staining were counted. The numbers at various BJ concentrations were the average of at least three different fields. The data were expressed as mean averages of three individual experiments (**P < 0.01). (C) Dose-dependent decrease of stained nucleus mitotic index PCNA. The tumor sections were incubated with rabbit antibody against PCNA followed by FITC-conjugated secondary antibody. The slides with PCNA fluorescence (green) were counterstained with DAPI (blue) before being analyzed confocal microscopy (scale bar, 50 μm). The images in the rectangular inset of tumor sections in mice treated with water are zoomed in at the top of the panel (scale bar, 15 μm). (D) BJ decreased nucleus PCNA signals. The number of fluorescent nucleus PCNA-positive cells in each field containing 100 cells as marked by DAPI staining was counted. The numbers of various BJ concentrations were the averages of at least three different fields. The data were expressed as mean averages of three individual experiments (*P < 0.05, **P < 0.01).