Additional Supporting Information may be found in the online version of this album.

HEP_25868_sm_SuppFig1.tif2656KSupporting Information Figure 1. Abnormalities in hepatic functions in DEN-treated Alb/AEG-1 mice. Serum levels of aspartate aminotransferase (AST) (A), alanine aminotransferase (ALT) (B) and alkaline phosphatase (Alk Phos) (C) in WT (n = 11) and Alb/AEG-1 (n = 17) mice were analyzed. The data represent mean ± SEM. *: p<0.01.
HEP_25868_sm_SuppFig2.tif2471KSupporting Information Figure 2. Alb/AEG-1 mice develop more aggressive tumors at 32 weeks when compared to WT mice. Photomicrograph of livers of mice (WT: n = 7; Alb/AEG-1: n = 9) treated with DEN at 32 weeks of age. The nodules in Alb/AEG-1 mice are much larger than those of WT mice. The asterisk indicates a markedly large nodule in Alb/AEG-1 mice.
HEP_25868_sm_SuppFig3.tif6607KSupporting Information Figure 3. Supervised samples and genes cluster analysis of genes whose expression is altered in DEN-treated Alb/AEG-1 livers when compared to DEN-treated WT livers. RNA was analyzed using Affymetrix oligonucleotide microarrays. Two-dimensional hierarchical clustering of samples and genes was performed using Pearson (centered) correlation and average linkage, based on the 25 probe sets that were significantly different between DEN-treated WT and Alb/AEG-1 livers. Each of the 25 rows in the heat map located beneath the dendrogram shows the relative expression for that specific probe set in the separate 6 samples (columns). The relative probe set expression levels are plotted according to the color scale at the bottom of the heat map, where red and green areas correspond to probe sets overexpressed and underexpressed, respectively, compared to the median intensity across the 6 samples.
HEP_25868_sm_SuppFig4.tif12950KSupporting Information Figure 4. Gene expression changes in DEN-treated Alb/AEG-1 mice. A. Analysis of mRNA levels of the indicated genes by real-time PCR using five animals/group. The most robust induction was observed for α-feto protein (Afp), a known marker for HCC (13), and for Tspan8, tetraspanin 8, which mediates tumor invasion, angiogenesis and metastasis (14). Tspan8 has also been identified as an AEG-1-induced gene in in vitro systems using human HCC cells and it is overexpressed in human HCC indicating that it might play an important role in mediating AEG-1-induced hepatocarcinogenesis (5, 15). Scd2, encoding stearoyl co-A desaturase, a lipogenic enzyme necessary for synthesis of monounsaturated fatty acids; Lpl, encoding lipoprotein lipase; and Apoa4 and Apoc2, encoding apolipoproteins A4 and C2, respectively, might play significant role in conferring the steatotic phenotype observed in Alb/AEG-1 mice. Lcn2, encoding lipocalin 2, promotes invasion and metastasis and is induced by AEG-1 (16). Tff3, encoding trefoil factor 3, a pro-angiogenic factor promoting invasion and metastasis and also overexpressed in HCC, is induced by AEG-1 (17, 18). In contrast, Meox2, a tumor suppressor gene with potent anti-angiogenic properties, is inhibited by AEG-1 (19). The Y-axis represents fold change in mRNA level when the expression level in WT mice was regarded as 1. The data represent mean ± SEM. *: p<0.01. B. Immunohistochemical analysis of the indicated proteins in the sections of livers from WT and Alb/AEG-1 mice.
HEP_25868_sm_SuppFig5.tif907KSupporting Information Figure 5. AEG-1 is overexpressed in hepatocytes isolated from Alb/AEG-1 mice. AEG-1 expression was detected by Western blot analysis using hepatocytes isolated from WT and Alb/AEG-1 mice with anti-AEG-1 and anti-HA antibodies. ?-tubulin was used as loading control.
HEP_25868_sm_SuppFig6.tif1635KSupporting Information Figure 6. AEG-1 protects hepatocytes from senescence. A. Analysis of senescence-associated markers in WT and Alb/AEG-1 hepatocytes. Cathepsin D and eEF1A1, two senescence-associated markers (20), were significantly upregulated and downregulated, respectively, in WT hepatocytes versus Alb/AEG-1 hepatocytes. B. Primary human hepatocytes were transduced with a lentivirus expressing GFP (Lenti.GFP) or expressing AEG-1 (Lenti.AEG-1). At day 6, Lenti.GFP-transduced primary human hepatocytes were vacuolated and >60% cells stained positive for SA ?-gal while Lenti.AEG-1 transduced cells were viable and none of the cells were SA ?-gal positive.
HEP_25868_sm_SuppFig7.tif728KSupporting Information Figure 7. TFF3 and FXII are efficiently knocked down by the corresponding siRNAs in Alb/AEG-1 hepatocytes. Alb/AEG-1 hepatocytes were either mock transfected or transfected with either control siRNA (siCon) or with siRNA for TFF3 or FXII. TFF3 (A) and FXII (B) mRNA levels were detected by real-time PCR and normalized by GAPDH mRNA level. The data represents mean ± SEM. *: p<0.05.
HEP_25868_sm_SuppFig8.tif491KSupporting Information Figure 8. AEG-1 does not change mature miR-181a level. miR-181a level was determined by real-time PCR in WT and Alb/AEG-1 hepatocytes obtained from four independent pairs of animals. The Y-axis represents fold change in miR-181a level when the expression level in WT hepatocytes was regarded as 1.
HEP_25868_sm_SuppFig9.tif562KSupporting Information Figure 9. Overexpressed AEG-1 is monoubiquitinated. Hep-pc-4 (control HepG3 clone) and Hep-AEG-1-14 cells (HepG3 cells stably overexpressing AEG-1) were treated or not with MG-132. The lysates were subjected to immunoprecipitation using anti-ubiquitin antibody and immunoblotting using anti-AEG-1 antibody. Five per cent of total cell lysate was used as input.
HEP_25868_sm_SuppFig10.tif397KSupporting Information Figure 10. Enzymes regulating fatty acid synthesis are overexpressed in Alb/AEG-1 mice. Lysates from WT and Alb/AEG-1 livers were subjected to Western blot analysis for the indicated proteins.
HEP_25868_sm_SuppTab1.doc42KSupporting Information Table 1. Fold-changes in gene expression in Alb/AEG-1 liver when compared to that in WT liver after DEN treatment. mRNAs that show at least two-fold change in Alb/AEG-1 hepatocytes compared to WT hepatocytes are displayed.
HEP_25868_sm_SuppTab1.xls40KSupporting Information Table 2. Fold-changes in protein expression in CM from Alb/AEG-1 hepatocytes when compared to that from WT hepatocytes. Proteins that are represented by at least two unique peptides and that show at least two-fold increase in Alb/AEG-1 hepatocytes compared to WT hepatocytes are displayed. The components of the coagulation pathway are highlighted in red.
HEP_25868_sm_SuppInfo.doc126KSupporting Information

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