Fig. 1: Overexpression of synNotch and its relationship to ligand-independent activation (LIA).

a Diagram of antigen-induced synNotch signaling. b Cells expressing PGK driven synNotch always express PGK driven mCherry. The matching promoter (Pro) drives the expression of a short-lived version of EGFP (d2EGFP). c Co-transfection efficiency measured by mCherry fluorescence. A total amount of 450 ng PGK driven plasmids includes a constant amount of 200 ng PGK driven mCherry and various amounts of PGK driven synNotch. One-way ANOVA for p values. Details on flow cytometry gating in Supplementary Fig. 1. d Diagram of synNotch LIA. LaG16 is the nanobody against GFP. tTAA is the transcription factor that induces d2EGFP expression downstream of TRE3G. e For experiments as in d, stacked histograms show the expression of d2EGFP when different amounts of PGK driven synNotch were transfected. For 0 ng synNotch, 250 ng plasmids that only contain the PGK promoter were used. f For experiments in e, the median d2EGFP fluorescence intensity of each co-transfection was calculated and presented as a scatter dot plot (bar:mean ± SD). Dashed line, 0 ng synNotch. g Cells were co-transfected similar to e, f but without TRE3G driven d2EGFP, fixed and stained (without permeabilization) with antibody against Myc tag. We inserted a Myc tag between the signal peptide and scFv to facilitate the detection of synNotch expression on the cell membrane. The fluorescence signal for mCherry and extracelluar Myc tag in different conditions are shown. The numbers on the upper right corners of each contour plot are the percentages of Myc-tag positive cells. h Cells stably expressing synNotch as in d were fixed and stained similar to g. i The sender cells with the corresponding antigen were incubated with cells expressing the synNotch as in d. j For experiments as in i, fold activation was calculated based on the mean values from synNotch cells co-cultured with sender cells with or without antigen. Two-tailed t-test for p values. k Similar to d, cells were co-transfected with synNotch with an EGF repeat inserted between LaG16 and NRR. l For experiments in k, EGF(−) stands for the synNotch in d, while EGF(+) stands for the synNotch in k. Two-tailed t-test for p values.