Immunocytochemical analysis and western blotting to detect LC3 protein

To evaluate the expression of LC3, the cells were fixed with 4% paraformaldehyde. The fixed cells were stained with anti-LC3 antibodies (M152-3; MBL Co. Ltd., Nagoya, Japan). Labeling was performed with the Zenon® labeling kit (Molecular Probes, Inc., Eugene, OR, USA). A fluorescence microscope (BZ-8000; Keyence, Osaka, Japan) was used for observation.

Total protein was extracted and quantified with a protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies for LC3 and β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA) followed with horseradish peroxidase-conjugated anti-mouse IgG (Promega, Madison, WI, USA), and enhanced chemiluminescence (ECL plus; Amersham Life Science, Amersham, UK), as per the manufacturer’s instructions.