Fig. 5.

Increased AMPK phosphorylation in cells overexpressing Hpa2. (A) Lysate samples of control (Vo) and Hpa2 overexpressing MKN-45 (left panels), BGC-823 (middle panels), and SGC-7901 (right panels) cells were subjected to immunoblotting applying the indicated antibody. (B) Immunofluorescent staining. Control (Vo) and Hpa2 overexpressing SGC-7901 cells were treated with vehicle (Con) or MG132 (20 µM) for 16 hours. Cells were then fixed with cold methanol for 10 minutes and subjected to immunofluorescent staining with anti-phospho-ACC antibody (red). Shown are representative images together with nuclear counter-staining (blue). Note stronger staining in Hpa2 cells before (upper right) and after MG132 treatment (lower right) vs control (Vo) cells. (C) Increased ACC phosphorylation by Hpa2 cells is HS-dependent. Hpa2 overexpressing SGC-7901 cells were left untreated or were treated with heparin (20 µM) added to the culture medium. Cell extracts were then prepared and subjected to immunoblotting applying anti-phospho-ACC (upper panel) and anti-ACC (lower panel) antibodies. (D, E) AMPK phosphorylation is decreased in gastric cancer. A tissue array that includes biopsies of gastric carcinoma tumors (T) and adjacent normal gastric tissue (N) was subjected to immunostaining applying anti-phospho-AMPK antibody (D). Higher magnification of the staining in normal and tumor biopsies is shown in (E). Note decreased AMPK phosphorylation in tumor samples vs normal gastric tissue (see also Table 3). AMPK, activated protein kinase; Hpa2, heparanase 2.