v-ATPase ^RNAi induces ISC/EB overproliferation. (a) Drosophila ISCs divide asymmetrically to self-renew and generate EBs. EBs differentiate to either ECs or enteroendocrine cells (EECs). Esg is expressed in ISCs and EBs; the Notch reporter NRE marks EBs. (b) Depletion of either Vha100-2 or Notch in ISCs by esg-GAL4 induced overproliferation of esg⁺ ISC/EB-like cells in the expanse of ECs in the posterior midgut (arrowheads). v-ATPase seems to promote Notch signaling in stem cell lineages, resulting in distinct consequences for ISCs and neural stem cells. Green represents esg-GAL4, UAS-GFP visualized by anti-GFP staining. (c) Percentage of esg⁺ ISC/EB-like cells per posterior midgut image. n (mCherry^RNAi) = 9, n (Vha100-2^RNAi)=12, n (Notch^RNAi) = 8. (d) Depletion of either Vha100-2 or Notch by esg-GAL4 results in reduced expression levels of the Notch reporter NRE-LacZ. NRE⁺ cells are marked by arrowheads. Green represents esg-GAL4, UAS-GFP visualized by anti-GFP staining. (e) Quantification of the percentage of NRE⁺ cells among esg⁺ cells per midgut image. n (mCherry^RNAi) = 9, n (Vha100-2^RNAi) = 9, n (Notch^RNAi) = 9. (f) Expression levels of v-ATPase subunits in sorted esg⁺ ISC/EB-like cells assessed by qRT-PCR upon depletion of Notch. n = 3 experiments. Error bars represent mean ± SD. ***, P < 0.001; ****, P < 0.0001; unpaired two-sided Student’s t test.