Whole-Cell Lysis and Western Blot Assay

Preparation of whole-cell cell lysates and Western blot analysis were performed as described previously (9 (link)). Briefly, cells were scraped and centrifuged at 4⁰C. Pellets were lysed in RIPA lysis buffer with 1 x Halt protease and phosphatase inhibitors cocktail (sc-364162, Santa Cruz Biotechnology). Protein concentrations were measured using Bio-Rad protein Assay (Bio-Rad). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to nitrocellulose membranes (0.45 µm, 1620115, Bio-Rad). Membranes were probed with specific primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega). Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate detection reagent (P90720, Sigma-Aldrich). Images were taken on the ChemiDoc XRS System (Bio-Rad) and the protein bands intensities were quantified by NIH ImageJ software. The relative values for various proteins regulated by autophagy induced by glucose starvation or metformin were calculated and shown as a ratio relative to control treatments.