Figure 7. GFP-LC3 processing can be used to monitor delivery of autophagosomal membranes. (A) Atg5^−/− MEFs engineered to express Atg5 under the control of the Tet-off promoter were grown in the presence of doxycyline (10 ng/ml) for one week to suppress autophagy. Cells were then cultured in the absence of drug for the indicated times, with or without a final 2 h starvation. Protein lysates were analyzed by western blot using anti-LC3 and anti-GFP antibodies. The positions of untagged and GFP-tagged LC3-I and LC3-II, and free GFP are indicated. This figure was modified from data previously published in reference 171, FEBS Letters, 580, Hosokawa N, Hara Y, Mizushima N, Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size, pp. 2623–2629, copyright 2006, with permission from Elsevier. (B) Differential role of unsaturating and saturating concentrations of lysosomal inhibitors on GFP-LC3 cleavage. HeLa cells stably transfected with GFP-LC3 were treated with various concentrations of chloroquine (CQ) for 6 h. Total lysates were prepared and subjected to immunoblot analysis. (C) CQ-induced free GFP fragments require classical autophagy machinery. Wild-type and Atg5^−/− MEFs were first infected with adenovirus GFP-LC3 (100 viral particles per cell) for 24 h. The cells were then either cultured in regular culture medium with or without CQ (10 µM), or subjected to starvation in EBSS buffer in the absence or presence of CQ for 6 h. Total lysates were prepared and subjected to immunoblot analysis. (B and C) are modified from the data previously published in reference 172.