2.6. GFP-LC3 or mCherry-GFP-LC3 assay

PC12 cells and primary neurons, PC12 cells infected with lentiviral shRNA to FLAG-BECN1 (S295A) or GFP, or PC12 cells infected with Ad-dn-Akt or Ad-LacZ, respectively, were infected with Ad-GFP-LC3 or Ad-mCherry-GFP-LC3 and seeded at a density of 5 × 10⁵ cells/well in a 6-well plate containing a PDL-coated glass coverslip per well. Next day, cells were treated with/without Cd (10 and/or 20 μM) for 8 h, 12 h and 24 h, or with/without Cd (10 μM) for 12 h following pretreatment with/without 3-MA (4 mM) for 1 h, with 5 replicates of each treatment. Afterwards, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4°C, and then washed 3 times with PBS. For the cells infected with Ad-mCherry-GFP-LC3, after treatments, the cells were stained by adding DAPI (4 μg/ml in deionized water) as described [39]. Finally, slides were mounted in glycerol/PBS (1:1, v/v) containing 2.5% 1,4-diazabiclo-(2,2,2)octane. All the cell images were obtained using a fluorescence microscope (Leica DMi8, Germany) equipped with a digital camera. For the cells infected with Ad-GFP-LC3, autophagosome formation was estimated by counting the number of the cells with GFP-LC3 (green). At least 50 cells were scored in each experiment. For the cells infected with Ad-mCherry-GFP-LC3, autophagosome and autolysome status was evaluated by counting cells with GFP⁺/mCherry⁺ (yellow) and GFP^–/Cherry-LC3⁺ (red) puncta, respectively. In tandem mCherry-GFP-LC3 assay, when an autophagosome fuses with the lysosome to form an autolysosome under acidic environments, GFP fluorescence is quenched in the autolysosome, whereas mCherry fluorescence is more stable. Therefore, when an autophagosome has not yet fused with a lysosome or when the degradation’s function of lysosome with acidification is impaired, co-localization of both GFP and mCherry fluorescence shows GFP⁺/mCherry⁺-LC3 (yellow) puncta in the merged image. In contrast, mCherry alone (without GFP) fluorescence presents GFP^–/mCherry⁺-LC3 (red) puncta, which corresponds to an autolysosome.