5-azacytidine (5azaC) and 4-phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich (St. Louis, MO). Preparation of cell

lysates, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoblotting analysis were performed as previously described.[13] Polyclonal-ADK (ab54818; Abcam, Cambridge, MA), monoclonal-ADK (F-5; Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (AC-15; Sigma-Aldrich) antibodies (Abs) were used. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect ADK messenger RNA (mRNA), as described previously,[14] using the primer sets (ADKF ZD1839 chemical structure and ADKR; ADK-5′-untranslated region [UTR]-187nts and ADK-5′-UTR checkR) listed in Supporting Table 1. Quantitative RT-PCR analysis for ADK mRNA was performed using

a real-time LightCycler PCR (Roche Diagnostics, PCI-32765 in vitro Indianapolis, IN), as described previously,[11] with the primer sets (ADKF and ADKR; ADK-5′UTR-384nts and ADK-5′UTR checkR; ADK-5′UTR-318nts and ADK-5′UTR checkR; ADK-5′UTR-187nts and ADK-5′UTR checkR; ADK-5′UTR-125nts and ADK-5′UTR checkR) listed in Supporting Table 1. RL assay was performed as described previously.[9] Experiments were performed at least in triplicate. Quantitative high-performance liquid chromatography (HPLC) analysis was performed using the extract from the OR6 or ORL8 cells treated with 50 µM of RBV for 8 hours. HPLC analysis was performed as described previously.[15] Small interfering RNA (siRNA) duplexes targeting the coding regions of human ADK (catalog no.: M-009687-01; Dharmacon, Inc., Lafayette, CO) were chemically synthesized. A nontargeting siRNA duplex (catalog no.: D-001206-13; Dharmacon) was also used as a control. ORL8 cells were transfected with the indicated siRNA duplexes using Oligofectamine (Invitrogen, Carlsbad, CA).[10] The methods of

plasmid construction for ectopic expression of ADK and MCE公司 retroviral infection using the constructed plasmids are described in the Supporting Materials. The method of plasmid construction for internal ribosome entry site (IRES) activity assay is described in the Supporting Materials. The dual luciferase reporter assay for IRES activity was performed by the method described previously.[14] Data are presented as means ± standard deviation. The Student unpaired t test was performed for statistical analysis between the two groups, and the difference was considered significant at P < 0.05. To identify the host factor responsible for the difference in RBV responses between Li23-derived ORL8 and HuH-7-derived OR6 cells, we first recompared the previous data from complementary DNA (cDNA) microarrays using Li23 and HuH-7 cells. Although we assigned 17 genes that showed dramatic differences in expression between Li23 and HuH-7 cells,[12] none of these genes were considered to be involved in the response to RBV.