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HEP_23026_sm_SupFig1.eps1958KSupplemental Figure 1. LA, BC, and CsA inhibited HCV RNA replication. OR6 cells were treated with none, LA (50 μM), BC (20 μM), and CsA (0.5 μg/ml) for 72 hours and subjected to Northern blot analysis using HCV specific RNA probe as previously described. β-actin was used as a control for the amount of RNA loaded per lane.
HEP_23026_sm_SupFig2AthroughC.eps1902KSupplemental Figure 2. Down regulation of MEK1/2 by siRNA attenuated the anti-HCV activity of LA but not PTV. (A) iMEK1 (Dharmacon; catalog no. M003571-01) and iMEK2 (Dharmacon; catalog no. M003573-03) were chemically synthesized siRNAs targeting MEK1 and MEK2, respectively. iGL2 (Dharmacon) as a control is the siRNA targeting luciferase GL2. OR6 cells were treated with indicated siRNA duplex using Oligofectamine (Invitrogen) and cultured for 24 hours and subjected to the treatment with PTV (0.5 μM) or LA (50 μM) for 72 hours. After the treatment, the RL assay was performed as described in the Materials and Methods section. Shown here is the relative luciferase activity (?) calculated when the RL activity of the control was assigned as 100%. The data indicate means+SDs of triplicate samples from at least three independent experiments. (B) Then, the ratio of the RL activity in the presence of the iMEK1 or iMEK2 to the RL activity of the control (iGL2) was calculated. (C) The production of p-ERK, MEK1/2 and β-actin in the cells treated with the reagents and siRNA were analyzed by immunoblotting as shown in the Materials and Methods section.
HEP_23026_sm_SupFig3AandB.eps927KSupplemental Figure 3. U0126 negated anti-HCV activity of CsA and LA, but not PTV. (A) Real-time LightCycler PCR was performed according to a method described previously.2 OR6 cells were treated with PTV (0.5 μM), CsA (0.5 μg/ml), and LA (50 μM) in the presence or in the absence of U0126 (10 μM) for 72 hours. (B) The ratio of HCV RNA in the presence of U0126 to HCV RNA in the absence of U0126 was calculated.
HEP_23026_sm_SupFig3C.eps1686KSupplemental Figure 3. U0126 negated anti-HCV activity of CsA and LA, but not PTV. (C) Immunofluorescence analysis was performed according to a method described previously. The OR6 cells were treated with the reagents for 96 hours as described above. The primary antibody used to detect core was anti-core (CP11; Institute of Immunology, Tokyo) and secondary antibody was Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch). The nuclear was stained with DAPI (Sigma). The cells were photographed under a confocal laser scanningmicroscope (LSM510; Carl Zeiss).
HEP_23026_sm_SupFig4.eps1962KSupplemental Figure 4. LA, CsA, and PTV inhibited HCV RNA replication in the serum free medium. OR6 cells were treated with LA (50 μM), CsA (0.5 μg/ml), and PTV (0.5 μM) in the serum free medium for 48 hours and then subjected to RL assay (upper panel) and Western blot analysis for core (lowere panel) as described in the Materials and Methods section.
HEP_23026_sm_SupFig5A.eps515KSupplemental Figure 5. NAC attenuated ERK phosporylation by AA, IFN-?, and CsA. (A) OR6 cells were pre-cultured as described in Figs. 4A and B, and then pre-treated with 10 mM NAC for 1 hour. Subsequently, the cells were treated with control medium, 100 μM AA, 2 IU/mL IFN-?, and 2 μg/mL CsA, respectively, in either the absence (?) or presence (+) of 10 mM NAC for 15 minutes. After the treatment, cell lysates were subjected to Western blot analysis as described in Fig. 5.
HEP_23026_sm_SupFig5BandC.eps3637KSupplemental Figure 5. NAC attenuated ERK phosporylation by AA, IFN-?, and CsA. (B) NAC negated CsA's anti-HCV activity. OR6 cells were pre-treated with 10 mM NAC for 1 hour. The cells were then treated with CsA (0, 0.25, and 0.5 μg/ml) with or without NAC (10 mM) for 72 hours. After the treatment, the RL assay was performed as described in the Materials and Methods section. Shown here is the relative luciferase activity (?) calculated when the RL activity of the control was assigned as 100%. The data indicate means+SDs of triplicate samples from at least three independent experiments. **?Significantly different from treatment with NAC (-) (**, p < .01). (C) Then, the ratio of the RL activity in the presence of the NAC to the RL activity in the absence of the NAC was calculated.
HEP_23026_sm_SupFig6.eps3722KSupplemental Figure 6. The effect of EGF on HCV RNA replication and cell growth in OR6 cells. OR6 cells were treated with EGF at 0, 50 and 100 ng/mL for 72 hours and subjected to an RL assay as described in the Materials and Methods section. For the cell growth assay, OR6 cells in 6-well plates were treated with EGF at 0, 50 and 100 ng/mL for 72 hours and subjected to trypan blue dye staining as described in the Materials and Methods section. *?Significantly different from treatment with control (*, p < .05).
HEP_23026_sm_SupFig7.eps1928KSupplemental Figure 7. Cell proliferation assay. The OR6 cells (2x103 cells) were plated onto the 96 well plate in triplicate at 24 hours before treatment of the reagents. The cells at 24 hours, 48 hours, and 72 hours after treatment were subjected to WST-1 cell proliferation assay (Takara Bio, Otsu, Japan) according to the manufacturer's protocol.

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