LC3 double-labeled lentivirus infection
We cultured 3×104 NRK-52E from different groups (control group (CON), rapamycin (RAP), rapamycin+Pink1-siRNA (RAP-PIN), myoglobin (100 μm), and myoglobin+Pink1-siRNA (MYO+PIN)) for 24 h in 24-well plates overnight. When the cell density was about 50%, 500 μl of HIGH-DMEM medium was added into a 1.5-ml centrifuge tube, together with 10 μl of mRFP-GFP-LC3 double-labeled lentivirus (the virus titer was 1×108 TU/ml, and MOI was 25), for 24-h incubation. After changing the medium, the incubation continued until the fluorescence infection efficiency was observed 72 h later. The degree of autophagy was calculated as RFP LC3+/number of total cells.