ICD of Notch ligands is required for Notch activity in the endogenous
system, but not in the aa-synNotch system. (A) Schematic of the Notch
luciferase activity assay. In this assay, sender cells that express
the Notch ligands Dll1 or Dll4 (hDll1/4) activate a Notch luciferase
reporter in the receiver cells. (B) Luciferase activity assay showing
the activation of a Notch reporter cell line (CHO-Notch1 transfected
with a 12xCSL-Luciferase reporter) co-cultured with CHO-TetR cells
expressing either wildtype hDll1/4 (hDll1/4), hDll1/4 lacking its
ICD (hDll1/4-ΔICD), or hDll1/4 with no lysine residues in its
ICD (hDll1/4-no lysine). (C) A schematic of the live-cell TEC assay
where inducible hDll1/4 (and its variants) are co-cultured with Notch1
fused to citrine in its NECD (Notch1-Citrine). (D) Images showing
a co-culture of inducible hDll1/4 variants (red) with Notch1-citrine
cells (green) 10 h after induction of ligand expression with 100 ng/mL
dox. While the full-length hDll1/4 exhibits strong TEC (white arrows),
both hDll1/4-ΔICD and hDll1/4-no lysine show accumulation on
the boundaries (orange arrows). (E) Schematic of an aa-synNotch system
with a receiver cell expressing an αGFP receptor, and a sender
cell expressing either a GFP ligand, or a GFP ligand with hDll4ICD
(GFP-ICD). (F) Luciferase activity assay with U2OS cells expressing
αGFP receptors co-cultured with U2OS cells expressing either
GFP or GFP-ICD ligands; (G) images showing co-culture of U2OS-GFP
or GFP-ICD ligands (red) with U2OS-αGFP-mCherry receptors (green).
Both ligands (GFP or GFP-ICD) show strong accumulation on the boundaries
between receiver and sender cells (orange arrows) and no TEC is observed.
Low levels of reverse TEC in the receiver cells are observed (blue
triangle). Data points show mean values from n =
7 for (B), and n = 20 for (F), from three and five
independent experiments, respectively. Error bars represent S.E.M.
**p < 0.01, ***p < 0.001.
Scale bars-10 μm.