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Figure S5. Accumulation of autophagosomes and ubiquitinated structures in Vps25-Vps34 mutant cells. In A–P, the eyeless-driven flippase technique (Stowers, R.S., and T.L. Schwarz. 1999. Genetics. 152:1631–1639) was used to generate eye discs comprised mostly of mutant tissue. (A–D) Toluidine blue–stained sections of mid-L3 eye-antenna discs (D) with part of the brain (B) also visible on the left side in each panel. Control (A) and Vps34 mutant (C) discs contain very few dying cells (dark blue spots). In contrast, numerous cells undergoing apoptosis are seen in Vps25 mutant discs (B), as previously reported (Herz, H.M., Z. Chen, H. Scherr, M. Lackey, C. Bolduc, and A. Bergmann. 2006. Development. 133:1871–1880), and also in Vps25-Vps34 double mutant discs (D). (E–L) Electron micrographs of disc cells. Ultrastructural analysis confirms the increased number of apoptotic cells in Vps25 mutant (F) and Vps25-Vps34 double mutant (H) discs relative to controls (E) and Vps34 mutants (G). Apoptotic cells can be recognized based on their progressively darkening cytoplasm and condensing nucleus. Numerous autophagosomes can be seen in dying Vps25 and Vps25-Vps34 double mutant cells. This is most obvious at an early stage of apoptosis when cytoplasmic darkening has started already, but the contents of autophagosomes remain relatively light (F and H, arrows), potentially because of being temporarily protected from caspase activity. The accumulation of autophagosomes (J and L, arrows) is also obvious in healthy Vps25 mutant (J) and Vps25-Vps34 double mutant (L) cells but not in controls (I) or Vps34 mutants (K). (M–P) Ubiquitin immunolabeling reveals the accumulation of ubiquitinated proteins in enlarged tubulovesicular structures in Vps25 mutant (M) and Vps25-Vps34 double mutant (O) cells. Autophagosomes do not show increased labeling in these cells (N and P, arrows). (Q and R) Confocal sections of eye discs containing Vps25-Vps34 double mutant clones. Ubiquitin-labeled structures (Q and R) colocalize with the endosomal marker Hrs (Q’) and are closely apposed to GFP-Atg8a punctae (R’). Yellow outlines indicate clonal boundaries. Arrows indicate structures positive for ubiquitin and Hrs (Q) or structures labeled for either ubiquitin or GFP-Atg8a (R). (S) Quantitation of autophagosome number in electron micrographs from control (A), Vps25^A3 (B), Vps34^∆m22 (C), and Vps25^A3 Vps34^∆m22 (D) imaginal disc cells. The number of autophagosomes in B and D is significantly greater than in A or C (P < 0.05; student’s t test). Error bars show SD from the mean. Genotypes: (A, E, and I) FRT42D/FRT42D GMR-Hid l(2)cell lethal; ey-Gal4 UAS-Flp/+, (B, F, J, M, and N) FRT42D Vps25^A3/FRT42D GMR-Hid l(2)cell lethal; ey-Gal4, UAS-Flp/+, (C, G, and K) FRT42D Vps34^∆m22/FRT42D GMR-Hid l(2)cell lethal; ey-Gal4 UAS-Flp/+, (D, H, L, O, and P) FRT42D Vps25^A3 Vps34^∆m22/FRT42D GMR-Hid l(2)cell lethal; ey-Gal4 UAS-Flp/+, and (Q and R) hsflp; FRT42D Vps25^A3 Vps34^∆m22/Cg-GAL4 FRT42D UAS-myrRFP.