Quantification of Autophagy Levels in Yeast

The YOM38 yeast cell containing the pR316-GFP-Atg8 plasmid yeast strain in glucose liquid medium with initial OD₆₀₀ value of 0.1 was treated with 0, 0.3, 1, and 3 µM GENI or 300 µM RES for 22 h. The yeast cells of different groups were collected and sonicated for 5 min. Cell lysates were centrifuged at 12,000× g for 15 min to obtain the supernatants for Western blot. Protein concentrations were measured using a BCA protein assay kit (CoWin Biotech, Beijing, China). Western blot was performed as described in our previous study [20 (link)]. Approximately 20 µg protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto poly (vinylidene fluoride) membranes. Membranes were incubated with primary antibodies followed by secondary antibodies. The primary antibodies used were as follows: anti-GFP (Medical & Biological Laboratories, Nagoya, Japan) and anti-β-actin (CoWin Biotech, Beijing, China). The secondary antibodies used were horseradish peroxidase-linked antirabbit IgGs (CoWin Biotech, Beijing, China). Antigens were visualized using an eECL Western Blot Kit (CoWin Biotech, Beijing, China) and digitized using the ImageJ software (National Institute of Health, Rockville, MD, USA).