Hela cells were seeded in 24-well plates and transfected with an RFP-GFP-LC3 adenoviral vector following the manufacturer’s protocol (Lianfeng Biological Company, Shanghai, China). The GFP signal is sensitive to acidic conditions and its fluorescence is quenched in the lysosomal lumen. Thus, high red fluorescence signal indicates a higher autophagic flux. After 24 hours of transfection, the cells were randomly divided into four groups: control group, HCQ group, ALA-PDT group, and ALA-PDT + HCQ group. Following a 6-hour treatment period, autophagosomes were observed using a fluorescence microscope (Nikon Eclipse E600, Japan). The quantification of yellow and red dots was conducted using Image J software (Bio-Rad Laboratories, USA).
