2.8. Measurement of Autophagic Flux

ARPE-19 cells were cultured in 6-well plates at a density of 6 × 10⁴ cells per well and transiently transfected with mCherry-eGFP-LC3 plasmid expressing an RFP-GFP-LC3 construct using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. After 72 h of transfection, the cells were treated with Ro 25-6981 for 8 h and then fixed with 4% formalin. Hoechst 33342 staining was performed to determine the degree of A2E accumulation and cellular apoptosis induced by BL irradiation, and the slides were examined using a laser scanning microscope (Nikon, Tokyo, Japan). Additionally, LC3-II levels were measured in samples treated with Baf A1, Ro 25-6981, or both compounds simultaneously for 8 h. Quantification of LC3-II band density was performed using ImageJ software.