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Figure 5 | BMC Chemical Biology

Figure 5

From: Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus

Figure 5

The effect of 25-hydroxycholesterol and siRNA knockdown or gene overexpression on HCV RNA replication and translation. Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 or neo/NS3-3'/5.1 subgenomic replicons were transiently transfected with siRNA duplexes homologous to the genes of interest (A and B) or with expression plasmids for the genes of interest (C and D) in the absence or presence of 5 μM 25-hydroxycholesterol (25-HC) for 48 hours. After siRNA knockdown or overexpression of target genes, cell lysates were analyzed for luciferase activity (A and C) and western blot analysis of HCV non-structural proteins NS3 and NS5A as well as PROX1 (B and D). GL2 and GL3 siRNAs were used as controls for siRNA knockdown as described in the Methods. Mock refers to cells that were incubated with the transfection reagent but without siRNA or plasmid. As a positive control, cells were separately treated with 100 U/ml IFNγ. Values in (A) and (C) are the mean normalized relative light units (RLU) per mg/ml of protein ± standard deviation from triplicate samples. For the siRNA knockdown (A), an asterisk above a bar indicates a significant decrease in HCV replication compared to mock-transfected cells (GL2, PROX-1; p < 0.01) or untreated cells (IFNγ; p < 0.01) in the presence of 5 μM 25-HC. For the overexpression studies (C), an asterisk above a bar indicates a significant decrease in HCV replication compared to cells transfected with the backbone β-gal vector (NK4; p < 0.05) or the untreated cells (IFNγ; p < 0.01) when compared to the appropriate treatment condition (no treatment or 5 μM 25-HC treatment as indicated).

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