2.5. mCherry-GFP-LC3 Adenovirus Transfection and Autophagy Assay

Cells cultured in 24-well plates (1 × 105 cells/well) were transfected with mCherry-GFP-LC3 adenovirus at 40 MOI for 24 h. After transfection, the cells were incubated with fresh culture medium again for 24 h. The numbers of GFP and mCherry dots per cell were counted in three randomly selected fields under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). GFP degrades in an acidic environment, while mCherry does not. Thus, yellow spots (formed from the overlap of red and green) indicate autophagosomes, while red spots indicate autophagic lysosomes. If autophagy is activated, the red signal will dominate over yellow. If autophagy is suppressed, there will be more yellow signal than red signal.