Discrimination between Notch activation mechanisms in transient transfected and stable cell lines. (A) Dor RNAi reduces Dx-induced Notch signal, and the Notch signal arising from shrub knockdown, but not the basal Notch signal or that resulting from tsg101 knockdown. (B) Two different TRPML RNAi knockdowns reduce Dx-induced Notch signal but not basal Notch activity. Error bars, SEM, *** indicates P < 0.001 by two-tailed t test. Sample numbers are indicated in the figure. (C–J) Comparison of transiently transfected and S2-R+ stable Notch expressing cell line. Fold activation of Notch signal in S2-R+ cells measured by NRE-firefly luciferase assay with overexpression of Dx and VPS4^EQ (C and F) or with RNAi knockdown of ESCRT-I (D and G) or -III components (E and H). S2-R+ cells were transiently expressing Notch (C–E) or the pMT-Notch stable cell line was used (F–H). (I and J) Pharmacological analysis of Notch signaling pathways induced by Dx and VPS4^EQ (I) or RNAi knockdown of ESCRT complexes (J) in the stable pMT-Notch/S2-R+ cell line, treated with 10 μM BB-94 or 50 μM ML-SI1. ** and *** indicate P < 0.01 and 0.001, respectively by two-tailed t test. Error bars represent SEM; number of samples indicated in the figure.