SEARCH

SEARCH BY CITATION

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

FilenameFormatSizeDescription
HEP_26260_sm_SuppFig1.eps1252KSupporting Information Figure 1. Impact of Silibinin (SbN) on HCV replication and cell proliferation (A) Huh7-Lunet cells harboring a persistent Luc/neo replicon of gt1b (Con1) or 2a (JFH1) were treated for 44h with the indicated concentrations of SbN. Mean values and SD from a representative experiment (n=2). (B) Impact of SbN treatment on proliferation of Luc/neo Con1 or JFH1 cells, respectively. Data represent mean and SD of triplicate values of a representative experiment (n=2). (C) Replication efficiency of different HCV genotypes after SbN-treatment. Huh7-Lunet cells were transfected with the respective replicons of genotype 1b (Con1), 1a (H77S) and 2a (JFH1) and treated with the indicated concentrations of SbN. Mean and SD of one representative experiment (n=2). (D) Impact of SbN treatment on proliferation of mock transfected Huh7-Lunet cells. Data represent mean and SD of triplicate values of a representative experiment (n=2). Medium of SbN treated as well as untreated control cells contained DMSO in a total concentration of 1%. 1μg/ml SbN corresponds to a concentration of 2.1 μM. Note that the slight genotype independent reduction of HCV replication at higher SbN concentrations coincides with cytostatic effects of the drug.
HEP_26260_sm_SuppFig2.eps1061KSupporting Information Figure 2. Analysis of viral sequences in a SIL non-responding patient. (A) Course of HCV RNA levels [IU/ml serum] in patient B chronically infected with HCV. LTx indicates the time of liver transplantation (day0). (A) Course of HCV titers in patient B (HCV genotype 1a) who received 20mg/kg/day of Legalon SIL (i.v.) starting at day -7 prior to LTx, but showed no specific response to SIL therapy. White bars depict HCV RNA levels before liver transplantation. Grey bars indicate application of SIL and black bars show HCV RNA levels after SIL therapy. (E) Conserved mutations identified in patient B after LTx by direct sequencing of RT-PCR products at day -42 (1.1×107 IU/ml) and 17 (6.2×106 IU/ml).
HEP_26260_sm_SuppFig3.eps915KSupporting Information Figure 3. Replication fitness of chimeric replicons Replication fitness of chimeric replicons depicted in Figure 5D. R.l.u. are normalized to the 4h value. Mean and SD of triplicate values of one representative experiment is depicted (n=3).
HEP_26260_sm_SuppFig4.tif14476KSupporting Information Figure 4. SIL does not affect the subcellular distribution of NS4B or NS4B/NS5A colocalization. (A) Immunofluorescence analyses of Huh7-Lunet cells expressing the non-structural proteins NS3-5B of JFH1 or Con1, respectively. The pictures show representative samples untreated or treated with SIL and stained for the non-structural proteins 5A (red), 4B (green). (B) Colocalization-analysis of the non-structural proteins 4B and 5A. Depicted are the mean and SD of the Pearson correlation coefficient (Rr) of 20 analyzed cells per condition.
HEP_26260_sm_SuppFig5.tif18840KSupporting Information Figure 5. SIL does not affect the NS4B/NS3 colocalization. Immunofluorescence analyses of Huh7-Lunet cells expressing the non-structural proteins NS3-5B of JFH1 (upper panels), or Con1 (middle panels) or Con1 Q1914R (lower panels), respectively, in presence (SIL) or absence (wo) of SIL, as indicated. The pictures show representative samples stained for the non-structural proteins 3 (red, left row), 4B (green, middle row) and merged pictures of both channels. The lower right corner of the merged pictures shows a detailed view of the region indicated by a white box.
HEP_26260_sm_SuppFig6.tif24115KSupporting Information Figure 6. SIL does not affect the induction and distribution of PI4P. (A) Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity as shown in (B) using ImageJ analysis (IntDen read-out) in presence and absence of SIL. Bars indicate the mean of arbitrary units (AU) +/- SD of 30 NS5A positive cells analyzed per condition, relative to the mean intensity of mock transfected cells. (B) Huh7-Lunet T7 cells were transfected with empty vector (mock) or with plasmids encoding the NS3 to NS5B polyprotein of JFH-1, Con1 or Con1 Q1914R, in presence (SIL) or absence (wo) of SIL, as indicated. 24h post transfection NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and nuclear DNA was stained with DAPI (blue). Note that expression of the HCV polyprotein results in a strong increase in intracellular PI4P levels, compared to mock transfected cells, which is not changed upon SIL treatment.
HEP_26260_sm_SuppFig7.tif6212KSupporting Information Figure 7. SIL does not modulate the NS4B-induced membrane morphology in cells expressing JFH1 NS3-5B. Ultrastructural analysis of the morphology of the membranous web (MW). Lunet-T7 cells transfected with pTM plasmids coding for NS3 to NS5B of isolate JFH1 in presence and absence of SIL were analyzed by transmission electron microscopy.
HEP_26260_sm_SuppFig8.tif5574KSupporting Information Figure 8. Silibinin (SbN) does not modulate the membrane morphology in cells expressing JFH1 or Con1 NS3-5B. (A) Percentage of multi-membrane vesicles (MMV) in Lunet-T7 cells transfected with pTM plasmids encoding NS3-5B of HCV JFH1 or Con1, respectively, in presence and absence of SbN (n=60). (B) Ultrastructural analysis of the morphology of the membranous web (MW) in presence and absence of SbN upon expression of NS3-5B of isolate JFH1 (upper panels) or Con1 (lower panels).
HEP_26260_sm_SuppInfo.doc104KSupporting Information
HEP_26260_sm_SuppTabs1_3.doc171KSupporting Information Table 1_3.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.