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

Figure 6.

Figure 6

Minimal ff-synNotch systems require the ligand ICD. (A,D) Schematics of the minimal ff-synNotch systems where both ligand TM domain (A) and EGF (1–8) repeats (D) are changed as indicated. Receptors lack the EGF (24–36) repeats. (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 after 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 on the boundaries, but no TEC were observed in 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 < 0.05, **p < 0.01, ***p < 0.001. Scale bars-10 μm.