RNA was quantitated as described previously. RNA was isolated using Trizol (Invitrogen), according to the manufacturer’s instructions. Complementary DNA was produced from RNA using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Gene-specific primer-probe sets were designed by Applied Biosystems (Foster City, CA).
We used an Applied Biosystems 7900HT Fast Real-Time polymerase chain reaction (PCR) system Cell Cycle inhibitor for quantitation of gene products. Gene expression was calculated, relative to hypoxanthine phosphoribosyltransferase, according to Pfaffl and depicted as fold increase compared to FBS. Huh7.5 cells were washed extensively with OptiMEM (Gibco, Grand Island, NY) to remove albumin (ALB) present in serum. The last wash was collected to determine background levels of ALB. Cells were then kept in OptiMEM at 37°C for 6 hours, and samples were
taken every 2 hours. The amount of secreted ALB was determined using quantitative enzyme-linked immunosorbent assay (ELISA), as described previously. ALB secretion (calculated as ng albumin/hour/10 × 106 cells) was normalized to FBS between experiments and expressed as fold-increase compared to FBS. Cells were grown on poly-L-lysine-coated coverslips and cultured in either FBS or HS. Lipid droplets were stained with Bodipy 493/503 (Invitrogen), according to the supplier’s instructions. Quantity of neutral lipid staining was OSI-906 visualized using a conventional fluorescence microscope (Zeiss Axiovert200; Carl Zeiss, Göttingen, Germany) and quantitated using ImageJ software (National Institutes of Health, Bethesda, MD). Images were taken using identical microscope and exposure settings. Data were collected in three independent experiments, MCE with four to eight microscopic fields per condition. Lipoprotein analysis was performed as described previously, using size-exclusion chromatography (large particles elute first) combined with in-line triglyceride (TG) and cholesterol measurements. Sucrose density-gradient ultracentrifugation
was performed as previously described. Fractions of 0.5 mL each were collected from the top of the gradient, and the RNA titer in each fraction was determined by quantitative reverse-transcriptase PCR (qRT-PCR). Immunoprecipitation (IP) experiments were performed as previously described. Freshly collected tissue culture supernatants from infected cells were filtered through a 22-μm filter and placed in clean tissue culture plates (without cells) and kept at 37°C or 4°C. Samples were taken at the start of the incubations and then at 4- and 12-hour intervals. Viral RNA was extracted and quantitated as described above. For calculation of significance, all experiments consisted of a minimum of three independent replicates.