Immunocytochemistry staining for LC3. To determine the status of LC3 protein, cells were seeded on 12 mm Fisherbrand Coverglass for Growth (Fisher Scientific, Pittsburgh, PA, USA) and cultured in a 24-well plate overnight. Huh7 cells treated overnight with Bafilomycin A1 (200 nM, Wako Chemicals, Richmond, VA, USA) were used as positive controls for autophagy. Cells were fixed in 4% paraformaldehyde (in PBS, pH 8), quenched in 50 mM NH 4 Cl for 10 min, permeabilized in 0.1% Triton X-100/PBS for 10 min, and blocked in 1% BSA for 10 min. Cells were then incubated with 15 ml rabbit anti-LC3 polyclonal antibody for 60 min, followed by goat anti-rabbit IgG conjugated with Alexa Fluor 488 for 30 min. Cell nuclei were stained with 10 mg/ml Hoechst 33258 (Invitrogen). All steps were carried out at RT. Cover slips were washed in water, mounted in a drop of ProLong Gold antifade reagent (Invitrogen), and air-dried in the dark overnight. Cells were imaged with a 40x (NA = 0.75) objective on a Zeiss AxioVision microscope equipped with a Hamamatsu monochrome camera. For direct comparison of staining intensity, exposure time was kept constant at 89 msec.