Autophagic flux assay
GFP-RFP-LC3 adenoviruses (HANBIO, Shanghai, China) were utilized to monitor autophagic flux. Briefly, when the cells reached 60% confluence, GFP-RFP-LC3 adenoviruses were added to the culture medium. After 8 h of adenoviral infection, the culture medium was replaced, and the cells were further incubated for 24 h. Subsequently, the cells were fixed with 4% formaldehyde for 10 min. Fluorescence images were captured via a laser confocal microscope (Zeiss, Jena, Germany). Typically, an increased number of red puncta (representing autophagic lysosomes) compared with yellow puncta (formed by the overlap of red and green) indicates activated autophagy, whereas a greater number of yellow puncta relative to red puncta suggests autophagy suppression.