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

HEP_25891_sm_SuppFig1.tif2397KSupporting Information Figure 1. The expression of c-Myc upregulates GPC3 and accelerates HCC cell proliferation. A, Western blotting showed increased levels of GPC3 in HepG2 cells that were transfected with the c-Myc expression plasmid. β-actin was used as a loading control. B, HepG2 cells were seeded into 6-well plates and were then transfected with the pcDNA3.1 empty plasmid or the pcDNA3.1-c-Myc expression plasmid. The cells were next stained with antibodies against GPC3 (green) and c-Myc (red). GPC3 exhibited localization both at the cell surface and within the cytoplasm after transfection with the pcDNA3.1-c-Myc expression plasmid. C, Flow cytometric cell cycle analysis revealed that both the overexpression of GPC3 and c-Myc significantly induced more S phase accumulation of Huh-7 cells, which suggestes increased cell proliferation. Western blotting confirmed that the transient overexpression of c-Myc stimulates GPC3 expression. D, Flow cytometric cell cycle analysis demonstrated more cells G1 phase-arrested Huh-7 cells upon co-transfection with the c-Myc overexpression plasmid and the siRNA-GPC3 plasmid. Consistent with the G1 arrest, this treatment decreased the proliferation rate of the HCC cells. The western blotting results indicated that GPC3 expression was decreased in the co-transfected cells. Three independent experiments were performed. E, The characterization of the expression of GPC3, c-Myc or c-Myc/siRNA-GPC3 by HCC cell proliferation assays. The pcDNA3.1 empty plasmid, GPC3, c-Myc or c-Myc/siRNA-GPC3 plasmids were transfected into Huh-7 cells, and the MTT assay was performed. The cell growth rate was significantly increased in cells that were transfected with the GPC3 or c-Myc expression plasmids compared with those transfected with the pcDNA3.1 empty plasmid and c-Myc/siRNA-GPC3 plasmid, there was no significant difference between the cells transfected with the pcDNA3.1 empty plasmid and c-Myc/siRNA-GPC3 plasmid (*P < 0.05, #P < 0.05 at days 2 and 3). F, The effects of GPC3 knockdown on the apoptotic rate of c-Myc-overexpressing cells. We co-transfected EGFP/c-Myc/siRNA-GPC3 (1:2:2) into Huh-7 cells, isolated for GFP-positive cells, and assessed the alterations in cell cycle and apoptosis. The results show that the co-transfection of c-Myc and siRNA-GPC3 synergistically enhanced apoptosis compared with c-Myc-transfected cells and the empty plasmid -transfected cells alone.
HEP_25891_sm_SuppFig2.tif291KSupporting Information Figure 2. The following plasmid combinations were co-transfected into the Huh-7 or HepG2 cells: EGFP/c-Myc (1:2), EGFP/GPC3 (1:2), EGFP/c-Myc/siRNA-GPC3 (1:2:2). The GFP-positive cells were isolated, and analyzed for sub-G0/G1 (apoptosis peak), G1, S, G2-M. The result shows that cells co-transfected with EGFP/c-Myc/siRNA-GPC3 plasmid exhibited significantly increased apoptosis.
HEP_25891_sm_SuppTab1.doc22KSupporting Information Table 1. Correlation between c-Myc expression and the differentiation of HCC
HEP_25891_sm_SuppTab2.doc22KSupporting Information Table 2. Correlation between GPC3 expression and the differentiation of HCC
HEP_25891_sm_SuppInfo.doc33KSupporting Information

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