1. Volume 42, Issue 3, Pages 358-364 (March 2005)
Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression 
Geum-Youn Gwak, Jung-Hwan Yoon, Kang Mo Kim, Hyo-Suk Lee, Jin Wook Chung, Gregory J. Gores 
Journal of Hepatology 
Volume 42, Issue 3, Pages (March 2005)
DOI: /j.jhep
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Hypoxia stimulates HCC cell growth. Huh-BAT, HepG2 and SNU-475 cells were grown under standard culture condition (20% O2 and 5% CO2, at 37°C) or hypoxic culture condition (1% O2, 5% CO2 and 94% N2, at 37°C). At each indicated time point, an MTS assay was performed according to the manufacturer's instruction. Data were expressed as mean±SD of percent change of optical densities as compared to that of the time 0. *P<0.05, vs. normoxia.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Hypoxia increases the expression of HIF-1α and hexokinase II in HCC cells. Huh-BAT, HepG2 and SNU-475 cells were cultured in a hypoxic culture condition for the indicated time periods. Equivalent amounts of proteins were immunoblotted with anti-HIF-1α, anti-hexokinase I, anti-hexokinase II, anti-HSP70, anti-mcl-1 and anti-actin antibody.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Hypoxia induces hexokinase II expression in an HIF-1α-dependent mechanism. Huh-BAT, HepG2 and SNU-475 cells were incubated with HIF-1α inhibitor, YC-1, at a concentration of 0–100μM in a hypoxic state. Cells were lysed after 12h and immunoblot analysis was performed using anti-hexokinase II and anti-actin antibody.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Hexokinase II inhibitor suppresses HCC cell growth. Huh-BAT, HepG2 and SNU-475 cells were incubated with HIF-1α inhibitor, YC-1, or hexokinase II inhibitor, 3-bromopyruvate (3-BP), at a concentration of 0–100μM under standard or hypoxic culture condition. After 24h, an MTS assay was performed according to the manufacturer's instruction. Data were expressed as mean±SD of percent change of optical densities as compared to that of control (0μM). *P<0.05, vs. normoxia.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Hexokinase II inhibitor induces apoptotic cell death. (A) Huh-BAT cells were cultured with 3-bromopyruvate (100μM) for 0 (left) and 6h (right). Apoptosis was investigated by using the nuclear binding dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence microscopy (at ×400 magnification). (B) Huh-BAT cells were cultured with 3-bromopyruvate (100μM) for the indicated time periods. The LDH activity in culture media and cell lysate of either floating dead cells or adherent viable cells were measured by using CytoTox 96 Non-Radioactive Cytotoxicity Assay according to the manufacturer's instruction. Data were expressed as mean±SD of percent change of optical densities as compared to that of the time 0. *P<0.05, vs. the time 0. (C) Huh-BAT cells were preincubated for 1h in the presence or absence of the pancaspase inhibitor, zVAD-fmk (75μM). Cells were then treated with 3-bromopyruvate (100μM) for the indicated time periods. Apoptosis was quantitated by DAPI staining and fluorescence microscopy. Data were expressed as mean±SD from three individual experiments. *P<0.05, vs zVAD-fmk(−).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Hexokinase II inhibitor induces apoptotic cell death through the activation of mitochondrial apoptotic signaling cascades. (A) Huh-BAT cells were cultured with 3-bromopyruvate (100μM) for the indicated time periods. Mitochondrial and cytosolic extracts were isolated, and equivalent amounts of cytosolic protein were immunoblotted with anti-caspase 8, anti-bid, anti-cytochrome c, anti-Smac/DIABLO, anti-AIF, anti-caspase 3 and anti-actin antibody. (B) Huh-BAT cells grown on coverslips were treated with 3-bromopyruvate (100μM) for 0–60min. Cells were incubated with anti-AIF antibody followed by Texas red-conjugated secondary antibody staining, and examined by confocal microscopy (at ×400 magnification).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions