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hep26421-sup-0001-suppfig1.tif1286KSupporting Fig. 1. RBV caused a reduction of GTP and accumulation of IMP. HPLC analysis using the nucleotides extracted from the OR6 or ORL8 cells treated with/without 50 µM of RBV for 8 hours was performed. HPLC analysis was performed as described in the Supporting Materials and Methods. The retention time of each nucleotide was confirmed using the authentic nucleotide purchased. (A) OR6 cells without RBV treatment. (B) OR6 cells treated with RBV. (C) ORL8 cells without RBV treatment. (D) ORL8 cells treated with RBV. Experiments were done in triplicate.
hep26421-sup-0002-suppfig2.tif1208KSupporting Fig. 2. No effect of inosine on HCV RNA replication. The ORL8 cells were treated with inosine for 72 hours, and then an RL assay was performed. The RLU (%) calculated at each point, when the level of luciferase activity in non-treated cells was assigned to be 100%, is shown. Experiments were done in triplicate.
hep26421-sup-0003-suppfig3.tif1343KSupporting Fig. 3. ADK is a determining host factor for the anti-HCV activity of RBV. (A) The intracellular GTP level was reduced in RBV-treated OR6-ADK cells. The signal of GTP obtained by HPLC analysis using nucleotides extracted from the RBV-treated OR6 or ORL8 cells was quantified as described in the Supporting Materials and Methods. (B) Intracellular IMP level was extremely high in RBV-treated OR6-ADK cells. The signal of IMP obtained by HPLC analysis using nucleotides extracted from the RBV-treated OR6 or ORL8 cells was quantified as described in the Supporting Materials and Methods. (C) sOR cells stably overexpressing ADK (sOR-ADK) were prepared by retrovirus-mediated gene transfer. The expression level of ADK in the sOR-ADK cells was monitored by Western blot analysis. (D) The sOR-ADK cells were dramatically converted from an RBV-resistant phenotype to an RBV-sensitive phenotype. sOR-ADK cells were treated with RBV for 72 hours, and then an RL assay was performed as described in Supporting Fig. 2. (E) The ORL8-ADK cells became more sensitive to RBV. ORL8-ADK cells were treated with RBV for 72 hours, and then an RL assay was performed as described in Supporting Fig. 2. Experiments of (D, E) were done in triplicate.
hep26421-sup-0004-suppfig4.tif1268KSupporting Fig. 4. Suppression of ADK expression in OR6 cells was not due to the genetic variations or epigenetic alterations in the ADK gene promoter. (A) ORL8c cells were transfected with pGL4.10/ADKP(ORL8) or pGL4.10/ADKP(OR6). After 48 hours, a luciferase reporter assay was performed as described previously.10 Experiments were done in triplicate. (B) OR6 cells were treated with 5azaC (10 μM) and/or 4-PBA (1 mM) for 48 hours. Total RNAs prepared from the treated or non-treated OR6 cells were subjected to RT-PCR using the primer sets for ADK and GAPDH (Supporting Table 2). (C) ADK expression level was not increased in OR6 cells treated with 5azaC. OR6 cells were treated with 5 µM 5azaC for 6 days. The expression levels of ADK in the treated and non-treated cells were compared by Western blot analysis. Western blot analysis was performed as described in the Supporting Materials and Methods.
hep26421-sup-0005-suppfig5.tif1247KSupporting Fig. 5. Comparative analysis of the 3'UTR species of ADK mRNAs derived from OR6 and ORL8 cells. The ratios of four different 3'UTR species of ADK mRNA obtained by 3'RACE analysis in OR6 and ORL8 cells are presented. Four poly(A) additional signals (AAUAAA) are indicated by open circles containing the numbers of nucleotides from the terminal codon in the ORF of ADK mRNA. The first nucleotide position of seed sequence to each miRNA is indicated in the each box.
hep26421-sup-0006-suppfig6.tif1288KSupporting Fig. 6. Expression levels of ADK in various hepatoma or immortalized hepatocyte cell lines were compared by Western blot analysis. The expression levels of ADK in OR6, ORL8, HuH6, HepG2, HLE, HLF, PLC/PRF/5, PH5CH8, Li21, Li22, Li24, Hep3B, NKNT-3, and OUMS29 cells were compared by Western blot analysis. Western blot analysis was performed as described in the Supporting Materials and Methods.
hep26421-sup-0007-suppfig7.tif1313KSupporting Fig. 7. The long-form 5'UTR of ADK mRNA possessed IRES activity regardless of HCV RNA replication. A reporter assay for IRES activity was performed using genome-length HCV RNA replicating OL8 cells or the OL8c cured cells. The OL8 or OL8c cells were transfected with the plasmid constructed for the assay of IRES activity. After 48 hours, a dual luciferase assay was performed. The ratio of the RL activity to the FL activity was calculated. The relative value calculated at each sample, when the ratio in the control vector-transfected cells (-) was assigned to be 1, is presented. (A) IRES activity in the OL8 cells transfected with reporter plasmids having 5'UTRs of different lengths. (B) IRES activity in the OL8c cells transfected with reporter plasmids having 5'UTRs of different lengths. (C) IRES activity in the OL8 cells transfected with the reporter plasmid having the deleted forms of the 5'UTR. (D) IRES activity in the OL8c cells transfected with the reporter plasmid having the deleted forms of the 5'UTR. (E) The secondary structure of the identified IRES region was predicted by the method of CentroidFold (). Experiments of (A-D) were done in triplicate. *P<0.05.
hep26421-sup-0008-suppfig8.tif1215KSupporting Fig. 8. No effect of SNP (rs10824095) on the IRES activity. The ORL8c cells were transfected with the plasmid (Fig. 4A) constructed for the assay of IRES activity. After 48 hours, a dual luciferase assay was performed. The ratio of the RL activity to the FL activity was calculated. Experiments were done in triplicate.
hep26421-sup-0009-suppfig9.tif1235KSupporting Fig. 9. Adenosine did not act as a trigger for IRES activity. The ORL8 cells were treated with adenosine for 72 hours, and then the cells were harvested for Western blot analysis. Western blot analysis was performed as described in the Supporting Materials and Methods.
hep26421-sup-0010-suppfig10.tif1221KSupporting Fig. 10. miR-424 did not enhance the translation of ADK. The OR6 and ORL8 cells were transfected with the expression plasmid of miR-424 for 48 hours, and then the cells were harvested for Western blot analysis. Western blot analysis was performed as described in the Supporting Materials and Methods.
hep26421-sup-0011-supptab1.tif1238KSupporting Table 1 The expression levels of IMPDH1 and IMPDH2
hep26421-sup-0012-supptab2.tif1359KSupporting Table 2 Primers used for this study
hep26421-sup-0013-suppinfo.doc50KSupporting Information

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