Autophagy was induced in PC3 and U87MG cells by Akt KD. (A) EM images of PC3 (a–d) and U87MG (e–g) cells grown in the absence (a and f) or presence (b–e and g) of Dox-induced Akt123 KD for 5 d. Arrows, degradative autolysosomes. Double arrows, initial AVs. Arrowhead, phagophore isolation membrane. M, mitochondrion in an AV. Asterisks, glycogen particle clusters. Bars: (a, b, f, and g) 0.5 μm; (c and d) 200 nm; (e) 1 μm. (B) Quantification of the number of AVs per unit cytoplasmic area of 4.5 μm² (n ≥ 64) and the percentage of cytoplasmic area occupied by AV in randomly sampled cytoplasmic areas (n = 5 areas of >200 μm²) of PC3 and U87MG cells with and without Dox-induced shAkt123 expression. Error bars represent SEM. (C) Dox-induced Akt silencing caused degeneration in PC3 and U87MG tumors. (a) PC3 tumors expressing the control EGFP shRNA after 15 d of Dox treatment. The tumor cells contain large nuclei and nucleoli, some lipid droplets (asterisks), and are connected by cell junctions (arrowheads). (b–d) PC3 tumors expressing shAkt123 after 15 (b and c) or 10 d (d) of Dox treatment. (b) Cells and nuclei in these tumors often appear shrunken. Arrows, AVs. E, eosinophil. (c) Two AVs (arrows) found among dilated RER cisternae in a degenerating tumor cell. (d) Ultrathin cryosection with immunogold labeling of human LAMP1. Label occurs on lysosomes (arrow) and AVs (top inset). Some of the tumor cells also contain human LAMP1–positive dense bodies with a shape reminiscent of microautophagy (bottom inset; de Waal et al., 1986). The tumor cells have widened nuclear envelope and ER cisterns (asterisks), which contain small cytoplasmic islands (arrowheads). (e) U87MG tumor after 5 d of vehicle treatment. (f–h) U87MG-shAkt123 tumor after 5 d of Dox treatment. Arrows, AVs. (h) In some tumor samples, cells with glycogen clusters (asterisks) and glycogen-containing AVs occur. Bars: (a–c) 2 μm; (e and f) 1 μm; (g) 0.5 μm; (d and h) 200 nm.