Different mechanisms of Notch signal activation induced by ESCRT-I, II, and III-Knockdown. (A) Fold activation of Notch signal in S2 cells by NRE-firefly luciferase assay after RNAi knockdown of ESCRT complexes and Su(dx). (B) Notch signaling after double knockdown of ESCRT components combined with mastermind, shibire, Rab5, Rab7, Kuzbanian, and GlcT-1 RNAi. (C–E) ESCRT KD-induced Notch signal activity after cells treated with (C) 10 μM BB-94, metalloprotease inhibitor, (D) cholesterol-depletion by 1% MβCD, or (E) 50 μM ML-SI1, TRPML inhibitor. (F) Notch activation by overexpressing VPS4^EQ in S2 cells. (G) VPS4^EQ-induced Notch signal when cells treated with BB-94, MβCD, or ML-SI1. (H–J) In vivo analysis of Notch signal in wing discs. (H) Ectopic Notch signal activation and tumor-like phenotype induced in the posterior half of wing discs by engrailed-gal4 and UAS-tsg101 RNAi or UAS-shrub RNAi. Shrub KD-induced Notch signal and the overproliferation phenotype can be suppressed by in car¹ genetic background but TSG101 KD-induced phenotypes are not affected. (I) Fluorescence intensity of NRE-GFP reporter in RFP-positive ventral side of the wing discs, normalized by NRE-GFP intensity along D-V boundary in RFP-negative side. (J) The overproliferation phenotype quantified as a ratio between RFP-positive and -negative area in each wing disc. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively, by two-tailed Student's t test. Error bars are SEM and sample sizes are indicated in the figure.