After Huh-7.5.1 cells were seeded onto coverslips for 24 hours, they were infected with adenovirus (moi 50) for 72 hours. Cells were fixed in 4% v/v formaldehyde in PBS, then permeabilized and blocked in PBS containing 0.05% saponin (wash buffer) and 1% BSA (binding buffer). Blocking of cells stained with BODIPY 493/503 was done in the presence of 0.2 M glycine to reduce background fluorescence. Cells were probed with primary antibodies in binding buffer, then incubated with a secondary antibody, Hoechst dye (10.0 mg/ml), and BODIPY 493/503 (1.0 mg/ml; when indicated) diluted in PBS. Cells were mounted onto slides with an anti-fade solution and sealed with clear nail polish. The slides were then imaged using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) or an Olympus Fluoview FV1000 laser scanning confocal microscope (Olympus Corporation, Tokyo, Japan) [95] [96] [97] . Leica MM AF Software (Leica Microsystems) was used to count the number of LDs (green channel) in cells expressing Spn4A.RRLL(r) (n = 21) or Spn4A.RRLL(s) (n = 15) (cells positive in the red channel). LDs in cells treated with Ad-Empty (n = 23) were also enumerated. All quantified images were acquired using the same laser intensity and gain settings, and LDs were enumerated by applying the same threshold setting to each image.