We referred to them as OR6c/JRN 3-5B cells. Immunofluorescence and Confocal Microscopic Analysis Cells were fixed sellckchem in 3.6% formaldehyde in phosphate-buffered saline (PBS) and permeabilized in 0.1% Nonidet P-40 (NP-40) in PBS at room temperature as previously described [21]. Cells were incubated with anti-HCV Core antibody (CP-9 and CP-11 mixture; Institute of Immunology, Tokyo, Japan), anti-Myc-Tag antibody (PL14; Medical & Biological Laboratories, MBL, Nagoya, Japan), anti-Alix antibody (Covalab, Villeurbanne, France), and/or anti-FLAG polyclonal antibody (Sigma, St. Louis, MO) at a 1300 dilution in PBS containing 3% bovine serum albumin (BSA) at 37��C for 30 min.

Cells were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) or anti-Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch) at a 1300 dilution in PBS containing BSA at 37��C for 30 min. Lipid droplets and nuclei were stained with BODIPY 493/503 (Molecular Probes, Invitrogen) and DAPI (4��,6��-diamidino-2-phenylindole), respectively. Following extensive washing in PBS, cells were mounted on slides using a mounting media of 90% glycerin/10% PBS with 0.01% p-phenylenediamine added to reduce fading. Samples were viewed under a confocal laser-scanning microscope (LSM510; Zeiss, Jena, Germany). RNA Interference The following siRNAs were used: human TSG101 (siGENOME SMARTpool M-003549-01-0005 and 5��-CCUCCAGUCUUCUCUCGUCUU-3�� sense, 5��-GACGAGAGAAGACUGGAGGUU-3�� antisense), human Alix/PDCD6IP (siGENOME SMARTpool M-004233-02-0005), human Vps4B (siGENOME SMARTpool M-013119-02-0005), human CHMP4b (siGENOME SMARTpool M-018075-00-0005), and siGENOME Non-Targeting siRNA Pool#1 (D-001206-13-05) (Dharmacon, Thermo Fisher Scientific, Waltham, MA) as a control.

siRNAs (50 nM final concentration) were transiently transfected into either RSc cells [14]�C[16] or OR6 cells [17], [18] using Oligofectamine Drug_discovery (Invitrogen) according to the manufacturer’s instructions.