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.