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. 2010 Aug;24(8):3052–3065. doi: 10.1096/fj.09-144519

Figure 5.

Figure 5

Effect of changes in autophagic activity and in the membrane components of the autophagic-lysosomal fusion. A) APGs and lysosomes (Lys) isolated from livers of mice fed or starved for 6 h before isolation were labeled with anti-LC3 (+FITC) or anti-LAMP-2B (+Cy5), respectively. Values represent percentage of fusion events relative to the total number of particles in the field and are means + se of 4 different experiments. B) APGs and Lys isolated from cells previously incubated with MDC and LysoTracker, respectively, were treated with 10 mg/ml of trypsin at room temperature for 15 min or with 10 mg/ml of proteinase K (PK) at 4°C for 15 min. Samples were collected by centrifugation; further action of the proteases was inhibited at the end of the incubation by addition of a cocktail of protease inhibitors. Efficiency of the protease treatment and of the protease inhibitor cocktail was tested by immunoblot as shown in Supplemental Fig. S9. Treated fractions were subjected to in vitro fusion assay. Fusion efficiency is expressed as percentage of fusion events relative to total number of particles in field; values are means + se of 3 different experiments. C) Starved mouse APGs labeled as in A were incubated with LAMP-2B-labeled lysosomes (left panel) or Texas-red asialoglycoprotein labeled endosomes (right panel) in fusion buffer alone (none) or supplemented with indicated amounts of cytosol from the same animals. Where indicated, 1 mM ATP, GTP, or NEM was added to the cytosolic fraction. Fusion efficiency was expressed as in A. Values are means + se of 3 different experiments. D, E) APGs and Lys isolated from livers of mice starved for 6 h were subjected to treatment with methyl-β-cyclodextrin (M). Levels of cholesterol in treated and untreated fractions were measured (D). Fractions were labeled as in A, and fusion events were analyzed (E). Values are means + se of 3 different experiments. F) Fusion events between monodansylcadaverine-labeled APGs and LysoTracker-labeled Lys isolated from NIH-3T3 cells and subjected to the same treatments as in E. Values are means + se of 3 different experiments are shown. *P < 0.05.