Analysis of GFP-LC3 puncta formation

Stably-transduced HCT116/HCT116 Beclin-1 KI (DM; D133A/D146A), ATE1^+/+, and ATE1^−/− MEF cells expressing the GFP-LC3 gene were generated via infection with the pCT-Autophagosome-GFP lentivirus (UPCI Lentiviral Facility) and selected with puromycin. Cells were fixed in 2% paraformaldehyde for 10 min. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signaling), mounted, and visualized using an OlympusFluoview 1000 confocal microscope and the companion software FV10-ASW2.1 under a 63× oil immersion objective. GFP-LC3 puncta formation in HCT116 cells was quantified by counting at least 300 cells and plotted as mean ± SD of three independent experiments.