Fig. S1. Categorized phenotypic view of 2R MENE mutants. (A-L) Eye imaginal discs stained with phalloidin to detect cortical actin. Compared with WT (A), discs entirely mutant for complementation groups MENE (2R)-A-F (C-H) show disorganized tissue architecture, increased proliferation, actin upregulation, and no photoreceptor differentiation, all hallmarks of neoplastic TSG mutations. For description of the phenotype of other MENE mutants (B,K-L), see Table 2. Scale bar: 100 m.

Fig. S2. vps28 is as an endocytic neoplastic TSG that does not control Staufen localization. (A-B) Rescue of epithelial architecture and ubiquitin accumulation in vps28^D2 mutants by Vps28 expression. Mutant cells are marked by presence of GFP are stained with anti-ubiquitin; the neoplastic TSG phenotype and the ubiquitin accumulation of vps28^D2 mutants (A) is rescued by ectopic expression of Vps28 in mutant cell (B). Single ubiquitin channel is shown in A’-B’. (C-F) Vps28 is not required for anterior localization of Staufen at mid-oogenesis. Stage 10 egg chambers containing ESCRT-I and ESCRT-II mutant germline cells and expressing GFP-Staufen are stained with phalloidin to reveal egg chamber morphology. Similarly to control egg chamber (C), both vps28^l(2)k16503 (D) and vps28^D2 (E) mutant oocytes show tight anterior GFP-Staufen localization (E; arrowhead). By contrast, vps25 egg chambers display diffusion of GFP-Staufen towards the oocyte center (F). Scale bars: 10 m (A-B), 50 m (C-F).