Time course effect of saponin on HCV propagation.

<p>(A) Huh7.5 cells were infected with Jc1 for 4 h and then treated with 10 µg/ml of saponin for 24 h, 48 h, and 72 h, respectively. At the indicated time points, HCV protein levels were analyzed by immunoblot analysis using anti-NS3 and NS5A antibodies, respectively. (B) Intracellular HCV RNAs were quantified by qRT-PCR at the given time intervals. (C) Relative HCV infectivity was determined by FFU as described above. The results are representative of three independent experiments. Error bars indicate the standard deviation.</p

Saponin suppresses viral propagation of IFN-α resistant mutant HCV.

<p>(A) Verification of IFN-α resistance of the mutant HCV. Huh7.5 cells were infected with either adaptive mutant HCV or IFN-α resistant mutant HCV for 4 h. The cells were treated with increasing amounts of IFN-α for 24 h and then total cell lysates were immunoblotted with the indicated antibodies. β-actin was used as a loading control for the same amount of cell lysates. (B) Saponin suppresses the NS5A expression in cells infected with IFN-α resistant mutant HCV. Huh7.5 cells were infected with HCV as described in the figure legend to A and then treated with increasing amounts of saponin for 24 h. Total cell lysates were immunoblotted with the indicated antibodies. (C) Co-treatment of saponin and IFN-α synergistically inhibits HCV replication in cells infected with IFN-α resistant HCV as similar level as in cells infected with adaptive HCV. Huh7.5 cells were infected with HCV as described in the figure legend to A and then treated with either 30 U/ml of IFN-α or 20 µg/ml of saponin alone, or in combination with IFN-α and saponin as indicated. Total cell lysates were immunoblotted with the indicated antibodies. (D) Huh7.5 cells were infected with HCV and then treated with either IFN-α or saponin alone, or in combination with IFN-α and saponin as described in the figure legend to C. Intracellular HCV RNAs isolated from these cells were analyzed by qRT-PCR.</p

Saponin inhibits HCV propagation.

<p>(A) Saponin inhibits HCV protein expression in a dose-dependent manner. Huh7.5 cells infected with either HCV Jc1 or mock for 4 h were incubated with the selected amounts of saponin (2.5, 5, 10, and 20 µg/ml) for 24 h. Cells were harvested and then equal amounts of cell lysates were immunoblotted with anti-NS3 and NS5A antibodies, respectively. β-actin was used as a loading control for the same amount of cell lysates. (B) Relative viral protein levels in HCV-infected cells treated with the indicated dosage of saponin were normalized by β-actin. (C) Huh7.5 cells were infected with Jc1 for 4 h and then treated with the indicated amounts of saponin. At 24 h postinfection, intracellular HCV RNAs were analyzed by qRT-PCR using a primer targeting HCV 5′UTR-core. (D) Relative HCV infectivity was determined by focus-forming units (FFU) as we reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039366#pone.0039366-Lim1" target="_blank">[16]</a>. Error bars indicate the standard deviation. * indicates statistical significance (p<0.05) and ** indicates p<0.01 versus control.</p

Saponin inhibits HCV propagation by regulating SOCS2 gene.

<p>(A) Huh7.5 cells were treated with either negative or SOCS2 siRNAs. At 72 h after transfection, cell viability was analyzed by cytotoxicity assay. (B-D) Silencing of SOCS2 impairs inhibitory activity of saponin on HCV propagation. Huh7.5 cells were transfected with the indicated siRNAs. At 36 h after transfection, cells were infected with HCV Jc1 for 4 h and then treated with 10 µg/ml of saponin for 24 h. Total cell lysates were immunoblotted with the indicated antibodies (B). Both intracellular HCV RNAs (C) and extracellular HCV RNAs (D) isolated from the culture supernatant were quantified by qRT-PCR. (E, F) Overexpression of SOCS2 suppresses HCV replication. Huh7 cells were transfected with either empty vector or FLAG-tagged SOCS2 expression plasmid. At 24 h after transfection, cells were infected with HCV for 4 h. Cell lysates harvested at 48 h postinfection were immunoblotted with the indicated antibodies (E). (F) Huh7 cells were treated as described in the figure legend to E. Intracellular HCV RNAs were isolated and analyzed by qRT-PCR.</p

Saponin potentiates IFN-αinduced anti-HCV activity.

<p>(A) Genomic organization of JFH1-derived luciferase reporter construct (JFH1-Luc). (B) Saponin suppresses HCV reporter activity in a dose-dependent manner. Huh7.5 cells were electroporated with JFH1-Luc RNA. At 48 h after electroporation, cells were treated with increasing amounts of saponin for 24 h and then luciferase reporter activities were determined. Data represent the mean of three independent experiments. (C) Huh7.5 cells were electroporated with JFH1-Luc RNA and were treated with increasing amounts of saponin in the absence or presence of 50 U/ml of IFN-α for 24 h and then luciferase reporter activities were determined. (D) Huh7.5 cells were electroporated with JFH1-Luc RNA and then treated with both saponin and IFN-α as described in the legend to Fig. 4C and cell viability was determined by cytotoxicity assay. (E) HCV subgenomic replicon cells were treated with either IFN-α alone or in combination with IFN-α and saponin as indicated. Cell lysates harvested at 36 h postinfection were immunoblotted with anti-NS3 antibody.</p

Saponin inhibits HCV replication in Huh7 cells harboring subgenomic HCV replicon.

<p>(A) HCV protein expressions were decreased by saponin. Both IFN-cured and subgenomic replicon cells were either left untreated or treated with 25 and 50 µg/ml of saponin, respectively. At 24 h after saponin treatment, total cell lysates were immunoblotted with the indicated antibodies. β-actin was used as a loading control for the same amount of cell lysates. (B) Total cellular RNAs were extracted from HCV replicon cells treated with saponin and intracellular HCV RNAs were quantified by qRT-PCR. (C) Subgenomic replicon cells were left untreated or treated with 25 and 50 µg/ml of saponin, respectively. At 24 h after saponin treatment, cell viability was determined by cytotoxicity assay.</p