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

HEP_26188_sm_SuppFig1.tif608KSupporting Information Figure 1. Expression of HIF-2α and its role in liver cancer cell. (A) The proliferation capability of MHCC97H cells cultured under normoxic or hypoxic (1% O2) conditions. (B) Expression of HIFs in MHCC97H and SMMC-7721 cells in different oxygen concentrations. (C) Expression of HIF-1α in MHCC97H clones with differing HIF-2α levels. (D) SMMC-7721 cells were transfected with pcDNA3.1-HIF-2α, pT2sh-HIF-2α, or the corresponding empty vector, then selected for G418 or puromycin resistance to isolate stably transfected clones. Left graph showed HIF-2α mRNA levels in representative monoclonal cells, error bars indicated standard deviation (n=3,* p <.01, two-tailed test). Right image: HIF-2α protein levels in representative monoclonal cells. (E) Cell proliferation as measured by CCK-8 in SMMC-7721 representative monoclonal cells. Results were expressed as the relative absorbance normalized to the one measured at zero time point. Error bars indicated standard deviation (n=6,*p <.01, two-tailed test). (F) The gene-silencing effect of siRNA targeting HIF-1α. Error bars indicate standard deviations. (G) Proliferation assay of cells with different HIF-1α or/and HIF-2α levels, cultured under 1% O2 condition. In the figure, si denotes siRNA 7146 targeting HIF-1α, and control cells were transfected with siRNA containing a scrambled sequence. Error bars indicate standard deviations. *p <0.05. (H) Cell proliferation measured by Ki67 in MHCC97H clone. Quantification of the relative percentage of Ki67 positive cells were shown in right image. Error bars indicated standard deviation (n=6, two-tailed test).
HEP_26188_sm_SuppFig2.tif1298KSupporting Information Figure 2. HIF-2α expression in orthotropic xenografts. (A) HIF-2α mRNA expression was confirmed in tumors formed by MHCC97H monoclones. (B) CD31 staining in tumors.
HEP_26188_sm_SuppFig3.tif2427KSupporting Information Figure 3. HIF-2α augments apoptosis by inhibiting the expression of TFDP3 Classification of HIF-2α-bound genes identified by ChIP-on-chip assay based on the biological function according to the enrichment score. It should be noted that some genes may be represented in more than one classified group.(B) HIF-2α target genes identified by ChIP-on-chip were classified based on cellular component according to the enrichment score. (C) HIF-1α levels in MHCC97H cells, which were transiently transfected by siRNA, pcDNA3-HIF-2α expression vectors, or control sequences. (D) TFDP3 levels were measured in MHCC97H cells with different HIF-1α levels. (E) The RNA level of TFDP3 and HIF-2α were tested in MHCC97H cells which was transiently transfected with pcDNA3.1- HIF-2α, pcDNA3.1-TFDP3 or mock plasmid. (F) The mRNA levels of other members of DP family were confirmed in the MHCC97H cells which were transiently transfected with HIF-2α (pcDNA3.1- HIF-2α), TFDP3 (pcDNA3.1-TFDP3) or empty vector (pcDNA3.1). (G) Gene-silencing efficiency of siRNA targeting E2F1. Error bars indicate standard deviations. (H) Proliferation of HIF-2α-overexpressing MHCC97H clones with different levels of E2F1. Error bars indicate standard deviations. *p<0.05. (I) The expression levels of apoptosis genes in HIF-2α-overexpressing MHCC97H cells with different levels of E2F1.
HEP_26188_sm_SuppInfo.doc37KSupporting Information
HEP_26188_sm_SuppTabs.doc126KSupporting Information Tables

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