Figure 4.

Methods used to assess autophagic activity. (a) Western blot analysis of MTOR, p-MTOR and LC3A/B-II under basal and rapamycin-induced conditions in the presence and absence of the fusion inhibitor BAF. Treatment with 25 nM rapamycin decreased the level of phosphorylation of MTOR, indicating that MTOR was inhibited by rapamycin. Treatment with 25 nM rapamycin caused a greater relative increase in LC3A/B-II after 2 h BAF treatment as compared to basal conditions, indicating that autophagic flux had increased by factor of 2.15. (b) Quantification of western blot analysis of the ratio of p-MTOR:MTOR and LC3A/B-II relative to basal conditions. (C) Fluorescence microscopy analysis of MEF GFP-LC3B cells with 75 nM LysoTracker Red. Cells treated with 25 nM rapamycin showed a small increase in the number of autophagosomes whereas autolysosomes increased significantly. Following BAF treatment there was an increase in autophagosomes and a decrease in autolysosomes. Fluorescence micrographs were acquired in multiple z-planes to quantify the complete autophagosome, autolysosome and lysosome pool size. Images shown here are projections of the z-stack images. Scale bar: 20 µm.