Figure 2.

Calibration of system conditions for data collection. (A) Images of cells expressing the pHluorin-mKate2-LC3 tandem fluorescent reporter following DMSO, 100 nM rapamycin (Rapa) and 500 nM bafilomycin A₁ (Baf A1) treatment. WT LC3 represent wild type and LC3 ΔG was used as a negative control because this mutant cannot be lipidated for phagophore association. (B) Autophagosomes and (C) autolysosomes were quantified over 90 min before addition of bafilomycin A₁ and 60 min after. R₁ was calculated using the first 20 min of data following bafilomycin A₁ treatment. (D) R₁ rates are plotted as a function of bafilomycin A₁ concentration. At least 150–200 cells were imaged for all experiments.