fig1

Figure 1. GLI activation is required for the survival of Huh-7 cells. (A) Protein-protein interaction network of the SHH signaling pathway, stemness, and EMT-involved proteins was obtained from the STRING database and purified by Gephi software. This network shows the importance of GLI2 and its connections with other proteins; (B) Quantifying mRNA expression levels of the GLI2 gene involved in the SHH pathway were assessed in 3 available HCC cell lines (Hep-3B, HepG2, and Huh-7) and compared to healthy liver cells by qRT-PCR. Despite the remarkable expression of GLI in HepG2 and Huh7 cells, we selected Huh-7 cells for this study due to some technical concerns; (C) The impact of decoy ODN on the cell viability of Huh-7 cells was evaluated by the Neutral red uptake assay at three different concentrations (450, 850, and 1,250 nM). The optimum inhibitory effect of GLI-specific decoy ODN was on 850 nM (56.36% ± 3%) compared to control (99.39% ± 3%) and vehicle (89.08% ± 3%) groups (Vertical stars are representative of statistically significant data compared to the control group); (D) The transfection efficiency of 850 nM Cy3-decoy on Huh-7 cells at three different time points (6, 24, and 48 h) after transfection. Cy3-decoy was transfected to cells with X-tremeGENE HP DNA. GLI-specific decoy ODN and the nuclei of HuU7 cells were counterstained with Cy3-decoy and Hoechst, red and blue, respectively (scale bars, 100 μm). Quantification of transfected cells demonstrated the highest transfection efficiency at six hours post-transfection (68.38% ± 7%). Data are presented as the mean ± SD for three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. GLI: Glioma-associated oncogene; SHH: Sonic Hedgehog; EMT: epithelial-mesenchymal transition; HCC: hepatocellular carcinoma; ODN: oligodeoxynucleotides; SD: standard deviation.