v-ATPase is required for brat tumor progression. (a) Knockdown of either the v-ATPase or Notch in a brat^RNAi background results in reduced larval brain tumor volume compared with mCherry^RNAi control. Larval brains were stained for Mira. OL, optic lobe. (b) Quantification of the brain tumor volume upon knockdown of brat in the combination with mCherry^RNAi, Vha68-2^RNAi, Vha100-2^RNAi, or Notch^RNAi. Each data point represents the volume of one brain lobe. n (mCherry^RNAi) = 9, n (Vha68-2^RNAi) = 11, n (Vha100-2^RNAi) = 6, n (Notch^RNAi) = 8. Error bars represent mean ± SD. ****, P < 0.0001; unpaired two-sided Student’s t test. (c) Vha68-2^RNAi or Notch^RNAi slowed down brat tumor progression and increased the survival of adult flies compared with brat^RNAi, mCherry^RNAi control. mCherry^RNAi does not target the RFP that we used to label tumor cells. Pictures of real-time tumor metastasis burden; RFP signal is displayed in false colors (BRGBCMYW). (d) Kaplan–Meier plot showing the increased survival of adult flies upon Vha68-2^RNAi or Notch^RNAi compared with mCherry^RNAi in a brat^RNAi background. n = 10 flies per genotype. (e) Cartoon depicting transplantation of brat tumors into adult host flies and subsequent treatment of these flies. CNS, central nervous system. (f) Single injection of Baf-A1 slows down brat tumor progression and prolongs fly survival compared with vehicle control. Pictures of real-time tumor metastasis burden; RFP signal is displayed in false colors (BRGBCMYW). First detectable metastasis appeared 3 d later in Baf-A1–treated flies in comparison with nontreated flies. (g) RFP signal intensity quantification of whole flies; RFP signals of all flies on one picture were averaged. n = 3 experiments. (h) Kaplan–Meier plot showing the increased survival of Baf-A1–treated flies compared with vehicle-treated flies. n ≥ 9 flies per genotype. *, P < 0.05; **, P < 0.01; Mantel–Cox test.