HeLa cells stably expressing YFP-Parkin using the Flp-In system (Invitrogen) were creating according to the manufacturer's instructions and maintained in 300 μg/ml hygromycin (Sigma-Aldrich). Rat cortical neurons were isolated on embryonic day 18 and grown in neurobasal media supplemented with B-27, l-glutamine, and penicillin/streptomycin. All cell culture materials were obtained from Invitrogen and all chemicals were obtained from Sigma-Aldrich. Chemicals were prepared from DMSO stock solutions, except paraquat, N-acetyl-cysteine, and 3-methyladenine, which were added fresh to media. Mfn1−/−,Mfn2−/− and Mfn1−/−,Mfn2−/− double knockout MEFs were generously donated by D.C. Chan (California Institute of Technology, Pasadena, CA), ATG5−/− MEFs were donated by N. Mizushima (Tokyo Medical and Dental University, Tokyo, Japan), Flp-In HeLa cells were donated by V.V. Lobanenkov (National Institutes of Health, Rockville, MD), and HeLa cells stably expressing GFP-LC3 were donated by A. Tolkovsky (Cambridge University, Cambridge, UK).