Analysis of Autophagy after GFP-LC3 Transfection

GFP-LC3 puncta formation was examined to show autophagy in RPTC cells as described previously.^8,13,40 Briefly, cells were plated on a coverslip to reach 50% confluence for transfection with 1 μg of GFP-LC3 by using Lipofectamine reagent (Invitrogen). The cells were then maintained in culture medium for 24 hours to reach 80%–90% confluence for cisplatin treatment. At the end of incubation, cells were fixed with 4% paraformaldehyde and examined by fluorescence microscopy to collect images and evaluate GFP-LC3 puncta.