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. 2022 Sep 15;11(10):3343–3353. doi: 10.1021/acssynbio.2c00247

Figure 5.

Figure 5

Removing the EGF repeats from the ff-synNotch ligands and receptors does not compensate for the lack of the ligand ICD. (A,D) Schematics of the ff-synNotch systems containing either ligands lacking the EGF (1–8) repeats in their ECD (A), or receptors lacking the EGF (24–36) repeats in their ECD (D). (B,E) Luciferase activity assay showing the activation of Notch reporter cell lines (U2OS cells stably expressing the receptors indicated in A,D and transfected with a luciferase reporter) co-cultured with CHO-TetR cells expressing different ligand variants corresponding to the constructs shown in (A,D). (C,F) Images from a TEC assay where U2OS cells expressing Notch-citrine variants were co-cultured with CHO-TetR cells expressing ligand variants corresponding to the constructs shown in (A,D). Images were taken 10 h induction of ligand expression with 100 ng/mL dox followed by 1 h induction of binding with 250 nM rapamycin. TEC was observed with full-length ligands (white arrows). Accumulation at the boundaries of cell contact without TEC was observed with ligands lacking their ICD (orange arrows). Data points show mean values from n = 14 measurements for (B,E) from four independent experiments. Error bars represent S.E.M. ***p < 0.001. Scale bars-10 μm.