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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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jws-hep.21004.fig1.TIF59K Nrf2 expression is increased in hepatocytes of rats fed with ethanol. Male Sprague-Dawley rats (150˜170g) were fed with a control diet or a diet containing ethanol (up to 35%) for 59 days. Hepatocytes were isolated from the liver by a collagenase perfusion. ( A ) Nrf2 and CYP2E1 proteins in the isolated hepatocytes were determined by Western blotting and quantified. ( B ) Nrf2 and GAPDH mRNA levels were determined by Northern blotting and quantified. The amount of Nrf2 mRNA was normalized to GAPDH mRNA. Results are expressed as arbitrary densitometric units or ratios under the representative protein or mRNA bands.&&num;160; The data are average &&num;177; S.E. (n=5). ***, p<0.001versuscontrol rats.
jws-hep.21004.fig2.TIF35K Nrf2 proteins is increased in E47 cells treated with ethanol. C34 and E47 cells (1&&num;215;10 6cells/well in 6-well plates) were treated with 100 mM ethanol for 0, 1, 2, and 3 days. After treatment, cells were collected and Nrf2 protein in the cell lysates was detected by Western blotting and quantified. Results are expressed as arbitrary densitometric units under the protein bands and represent average &&num;177; S.E. (n=3). *, p<0.05versuscontrol E47 cells without treatment.
jws-hep.21004.fig3.ppt77K SiRNA-Nrf2 decreases the mitochondrial membrane potential of E47 cells .C34 and E47 cells (2.5&&num;215;105 cells/well in 6-well plates) were transfected with SiRNA-Control or SiRNA-Nrf2 according to the method described in Materials and Methods. Thirty hours after transfection, mitochondrial membrane potential was detected by flow cytometry analysis using Rh123 and PI. Data presented here are one representative image of each group, and show the percent cells in the M1 low mitochondrial membrane potential zone.
jws-hep.21004.fig4.ppt37K Suppl Fig 4.SiRNA-Nrf2 increases ROS and decreases the viability of E47 cells .C34 and E47 cells (5&&num;215;10 4cells/well in 24-well plates) were transfected with SiRNA-Control or SiRNA-Nrf2 according to the method described in Materials and Methods. (A) ROS levels were measured at 0, 1, 2, and 3 days after transfection. Results are expressed as arbitrary units and the amount of ROS in C34 cells transfected with SiRNA-Control at day 0 was assigned a value of 1. (B) Cell viability was measured by MTT assay and calculated as percentage of viability compared to that of cells transfected with SiRNA-Control at day 0 (assigned as 100% viability). Results are expressed as average &&num;177; S.E. n=3; *, p<0.05; **, p<0.01, compared with the group transfected with SiRNA-Control at day 0.

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