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. 2015 Apr 23;22(11):1812–1823. doi: 10.1038/cdd.2015.34

Figure 4.

Figure 4

p32 regulates starvation-induced autophagy. (a) Protein extracts from Hela cells infected with p32-Myc lentiviral construct were analyzed by western blotting with the indicated antibodies. (b) Hela cells infected with the indicated lentiviral p32 shRNAs were collected, and cell lysates were subjected to western blotting with the indicated antibodies. (c) Hela cells stably expressing the indicated shRNAs were placed in normal or in EBSS starvation medium in the presence or absence of lysosomal protease inhibitors PE for 6 h. Lysates were immunoblotted as indicated (upper panel). p62 and LC3-II levels were quantified by densitometry and plotted as mean±S.D. of three independent experiments (middle and lower panel). (d) p32 knockdown Hela cells were untreated or treated with EBSS medium for 6 h. Cells were then fixed and analyzed by transmission electron microscopy (TEM). The bottom panels show enlarged views of the boxed areas. Quantification of the autophagic vacuoles (AVs) was shown as mean±S.D. of 15 random areas. **P<0.01. (e) H1299 cells stably expressing GFP-LC3 were infected with p32-shRNA and stably reconstituted with lentiviral Myc-ULK1 vector. Cells were then placed in EBSS medium for 6 h. GFP-LC3 positive autophagosomes were determined by confocal microscopy. The scale bar represents 20 μM (upper panel). Histogram represents the percentage of cells containing positive LC3 punctas. The error bars represent the mean±S.D. of five random areas (lower panel). **P<0.01. (f) Hela cells stably expressing the indicated shRNA-resistant forms of p32 were infected with p32-shRNA lentiviruses. Histogram represents the percentage of viable cells scored by Annexin V staining and FACS analysis after being cultured in EBSS medium for 6 h. The error bars represent the mean±S.D. of three independent experiments. ***P<0.001. (g) Hela cells were infected with p32-shRNA and stably reconstituted with lentiviral Myc-ULK1 vector. Histogram represents the percentage of viable cells scored by Annexin V staining and FACS analysis after being cultured in EBSS medium for 6 h. The error bars represent the mean±S.D. of three independent experiments. **P<0.01, ***P<0.001