Figure 1. Manipulation of endogenous NOTCH1 signaling in MCF10A cells.

(A) The NOTCH1 (N1) full length receptor (N1FL) is composed of the N1 extracellular domain (N1ECD) and of the N1 transmembrane domain (N1TM). The N1ECD contains several EGF-like repeats, some of which are binding sites for Notch ligands and the negative regulatory region, which includes the Lin12/Notch Repeats (LNR) and the heterodimerization domain (HD). The N1ECD and N1TM portions of Notch receptors are produced by Furin cleavage occurring at the S1 site during trafficking to the trans-Golgi network and are non-covalently held together by Ca2+ at the HD. Ca2+ depletion causes ADAM10 and γ-secretase to sequentially cleave the receptor at S2 and S3 sites. This leads to the release in the cytoplasm of the N1 intracellular domain (N1ICD), which translocates to the nucleus. N1ICD contains the RBPJ-kappa-associated module, ankyrin repeats (ANK), nuclear localization signals, and the proline (P), glutamic acid (E), serine (S), and threonine (T) – PEST domain, which limits NICD half-life. (B) Immunofluorescence reveals that EGTA treatment relocates most endogenous NOTCH1 from the cell surface to the nucleus and this is inhibited by silencing ADAM10 or PSENEN. (C) siRNA against ADAM10 or PSENEN effectively depletes their mRNA. (D) The Notch reporter cell line, MCF10A-RbpJk-Luc, reports strong EGTA-induced Notch signaling, and this effect is markedly suppressed by silencing NOTCH1, PSENEN, or ADAM10. (E) Western blot analysis of MCF10A cell extracts using an antibody against S3-cleaved N1 shows that unstimulated cells express low levels of N1ICD, which appears as a doublet. γ-secretase inhibition for 3 h (DAPT) markedly reduces basal levels of the N1ICD lower band. (F) Western blot analysis of MCF10A lysates indicates that λ-phosphatase (λ-PPA) treatment leads to disappearance of the upper band of the N1ICD doublet, indicating that it corresponds to a phosphorylated N1CD form. *** and **** indicate P < 0.001 and P < 0.0001, respectively.