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

FilenameFormatSizeDescription
HEP_23169_sm_SuppDoc.doc46KSupplementary Materials and Methods
HEP_23169_sm_SuppTable.doc51KSupplementary Table 1
HEP_23169_sm_SuppFig1.tif207KSupplementary Figure 1. Bcl-2 family proteins expression in cells harbouring HCV replicons. Protein extracts from parental (Huh7) and replicon expressing (3-5B and C-5B) cells were analyzed by immunoblotting for Bak, Bim, Bcl-2, Bcl-xL and Bax expression. GAPDH served as a loading control.
HEP_23169_sm_SuppFig2.tif156KSupplementary Figure 2. Pharmacological inhibition of proteasome or caspases has no effect on Bid expression. Parental (Huh7) and replicon expressing (3-5B and C-5B) cells were grown for 24 hrs in the absence or in the presence of proteasome (lactacystin, 50 μm) or caspase (z-VAD-fmk, 10 μm) inhibitors, as indicated. Protein extracts were analyzed for Bid expression by immunoblotting. GAPDH served as a loading control.
HEP_23169_sm_SuppFig3.tif112KSupplementary Fig.3 Pharmacological inhibition of proteasome or caspases has no effect on Bid stability. Recombinant human Bid (50 ng) was incubated with soluble extracts of parental (Huh7), full-length replicon (Nneo/C-5B) cell lines for 3h at room temperature. Proteasome (lactacystin, 50 μm), caspase (z-VAD-fmk, 10 μm) or calpain (MDL-28170, 100 μm) inhibitors were added, as indicated. rBid degradation was analysed by immunoblotting.
HEP_23169_sm_SuppFig4.tif207KSupplementary Fig. 4 Calpain expression is not modified by HCV proteins. (A) Calpain 1 and 2 protein expression in Huh7, Nneo/C-5B and Nneo/3-5B HCV cells was analysed by immunoblotting. GAPDH expression served as a loading control. (B) RT-qPCR analysis of calpains mRNA expression in the parental and replicon containing cells. Data are normalized to the expression of GAPDH. Only the calpains that gave a signal in the assay are shown.
HEP_23169_sm_SuppFig5.tif229KSupplementary Figure 5. Viral protein expression in stably transfected cell lines. Huh7 cells were transduced with single, or a combination of, retroviral vector(s) coding for the myc-tagged HCV1b proteins core, NS3, NS4A and NS5A, as indicated. Viral protein expression was revealed by immunoblotting with the 9E10 anti-myc monoclonal antibody.
HEP_23169_sm_SuppFig6.tif145KSupplementary Figure 6. Bcl-2 family proteins expression is unaltered in NS5A-expressing Huh7 cells. Protein extracts from parental (Huh7) and NS5A-expressing Huh7 (Huh7-NS5A) cells were analyzed for expression of Bak, Bim, Bcl-2, Bcl-xL and Bax by immunoblotting. GAPDH served as a loading control.
HEP_23169_sm_SuppFig7.tif95KSupplementary Figure 7. NS5A expression reduces apoptosis in TNF? treated cells. Parental and NS5A expressing Huh7 were pre-treated, as indicated, with MDL-28170 for 6 hours. TNFa (0.5 ng/ml) and actinomycin D (0.5 μ g/ml) were added for an additional 18 hours. Apoptosis was assayed by counting DAPI labelled nuclei. For each experimental condition at least 500 cells were analyzed in randomly chosen fields. Results are presented as mean+/- SEM of three separate experiments (*p<0.01,**p<0.001).
HEP_23169_sm_SuppFig8.tif5453KSupplementary Figure 8. Bid is efficiently downregulated by shRNA. Parental and NS5A expressing Huh7 cells were transduced with retroviral vectors encoding shRNA for Bid or for Firefly luciferase (luc). Protein extracts were analyzed for NS5A and Bid expression by immunoblotting. GAPDH served as a loading control.

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