FIGURE 4. Jo2 and TNF-α induce changes in the macroautophagic compartment.

A and B, MEFs treated with Jo2 (Fas), TNF-α, or menadione (Men) for 4 (A) or 24 h (B) were subjected to immunoblotting with an antibody for LC3. Where indicated, 20mm ammonium chloride and 100µm leupeptin (NH₄Cl/Leup) were added to the incubation medium. Stripped membranes were reprobed for β-actin. The unconjugated (I) and conjugated (II) LC3 forms are indicated. The LC3-II to LC3-I ratio (II:I) and the -fold increase in LC3-II in the presence of the protease inhibitors (+PI:None) are shown. Values are the means from densitometric scans of immunoblots from 3 independent experiments. Con, untreated. C and D, immunofluorescence staining for LC3 in the same cells. C, representative fields are shown (bar: 5µm). D, the numbers of fluorescent punctate structures per cell (left) and the average size of the LC3-positive puncta (right) were determined in 20 cells/experiment. Data are from 3 independent experiments (*, p < 0.05 between control and Fas or TNF-α treatment).