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Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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suppmat_fig1.tif1679KPKA activity in HepG2 cells treated with vitamin K2.(a)Protein cell lysates were prepared from HepG2 cells treated with 50 mM vitamin K2 for 0, 2, and 6 h; the lysates were tested for the ability to phosphorylate a PKA-specific peptide. The phosphorylation state of the peptide was examined by agarose gel electrophoresis. Under basal conditions, PKA activity is low. Treatment with vitamin K2 for 2 h induced PKA activation.(b)Protein cell lysates were prepared from HepG2 cells treated with 0, 50, 100, and 150 mM vitamin K2 for 2 h; lysates were tested for the ability to phosphorylate a PKA-specific peptide as described in (a). Treatment with 5 mM H89 in addition to 100 mM vitamin K2 attenuated the st imulatory effect induced by vitamin K2. nc, negative control.(c)Protein cell lysates were pre pared from HepG2 cells treated with 0, 20, 30, 40, 50 and 100 mM vitamin K2 for 2 h. PKA activity was tested as described in (a). nc, negative control.
suppmat_fig2abc.tif1145KVitamin K2 suppresses hepatocellular carcinoma cell growth in vivo.<br />Two weeks after the subcutaneous inoculation of athymic nude mice with 1 x 107 growing PRF/PLC/5 cells, animals were randomized to orally receive either vitamin K2 (20 mg/kg/d) or vehicle. The differences in body weight(a)and tumor volume(b)between the second and fourth weeks after randomization were divided by the values at the second week. Four and three animals were examined in the vehicle and vitamin K2 groups, respectively. * P < 0.05.(c)After a 6-week treatment, part of each tumor from vitamin-K2-treated mice (n = 2) and vehicle-treated mice (n = 2) was lysed in extraction buffer and assayed for PKA activity. nc, negative control.
suppmat_fig2d.tif3939KVitamin K2 suppresses hepatocellular carcinoma cell growth in vivo.(d)Representative cross-sectioned tumors stained with hematoxylin and eosin (40x and 200x magnification)
suppmat_fig3.tif1065KVitamin K2 does not influence on the Cdc25A activity. HepG2 cells were treated with 50 mM vitamin K2 for 0, 2, or 6 h. Cells were harvested, and subjected to SDS-PAGE. After transfer to a membrane, proteins were blotted with anti-Cdc25A, anti-cdc2, or anti-cdc2-phospho Tyr15 (cdc2-Y15P). Anti-cdc2-phospho Tyr15 reacts with phospho-Tyr15 in both cdk2 and cdc2 (both are 34 kDa). Non-phosphorylated cdc2 recombinant protein (32 kDa) and phosphorylated cdc2 protein (34 kDa) were used as negative and positive controls for the phospho-selectivity, respectively. The experiments were performed twice and similar results were obtained. Representative immunoblots are shown.

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