Fig 2: Cell-Autonomous Suppression of DLL1 Function by NOTCH2NLB(A and B) Co-immunoprecipitation of NOTCH2NLB-myc (N2NL-FL-myc) and DLL1-GFP in HEK293T cells, using anti-GFP (A) and anti-Myc antibody (B).(C) CHO cell line expressing DLL1 was transfected with a NOTCH2NLB expression plasmid. DLL1 protein at the plasma membrane was detected by NOTCH1-Fc (green in [C] and white arrows) and total DLL1 protein using DLL1 antibody (red in [C]). Anti-Myc antibody was used to identify NOTCH2NL-expressing cells (blue in [C], white arrowhead) among non-expressing cells (open arrowheads).(D and E) The fluorescent intensities of NOTCH1-Fc (revealing DLL1 protein at the cell plasmic membrane) and of DLL1 antibody (revealing DLL1 protein in the whole cell) were measured in NOTCH2NLB-expressing cells and non-expressing cells (plots of three independent experiments). Compared to the non-expressing cells, NOTCH2NLB full-length-expressing cells show lower level of NOTCH-Fc signal, while NOTCH2NL-?EGF or NICD expressing cells display same levels as control cells (D). The ratio of signals for NOTCH1-Fc over DLL1 was quantified for each individual experiment (E).(F–L) NOTCH2NLB suppresses DLL1 function in vivo. Mouse cortex in utero electroporation (E13.5, analysis at E15.5) was performed with DLL1 alone or together with N2NL-FL or N2NL-?EGF. Bin analysis reveals that the proportion of electroporated cells is decreased in VZ (J) and increased in CP (K) following DLL1 overexpression, which is blocked by NOTCH2NL-FL but not by NOTCH2NL-?EGF (F–K). The same results were obtained when examining the proportion of PAX6 progenitors in electroporated cells (F–I and L).Data are presented as mean ± SEM and p values by Student’s t test (B and C) and one-way ANOVA and Bonferroni post-hoc test (H, I, and N). Scale bars, 10 µm (B) and 100 µm (E and K). See also Figure S5.

Fig 3: Cis-activation is affected by changes in ligand-receptor affinity.(A) (Top) Cell lines used for analyzing effect of Rfng on cis-activation. (Bottom) Plots showing mean Notch activation (reporter Citrine fluorescence normalized to background fluorescence in uninduced cells) in N1D1 (black) or N1D1 + Rfng (purple) cells expressing different levels of Dll1 (measured using co-translational mCherry fluorescence). Error bars indicate s.e.m (n = 3 replicate experiments). (B) Heatmaps of mean Notch activation (n = 3 replicates), relative to background reporter fluorescence, in N1D1 + Rfng (upper panel) or N1D1 (lower panel) cells induced with different [4epi-Tc] (columns) and cultured at different relative fractions (rows). Upper panel is the same data in Figure 1—figure supplement 2C, replotted for direct comparison. Rfng expression predominantly affects signal amplitude (compare intensity scales). (C,D) (Top) Cell lines used for analyzing effect of ligand on cis-activation of Notch1 (C) or Notch2 (D). (Bottom) Comparison of mean cis-activation in polyclonal populations (‘Pop’) of cells co-expressing Dll1 or the higher affinity ligand Dll4 with the indicated receptor, as a function of ligand expression, read out by co-translated H2B-mCherry fluorescence. Values represent mean of 3 replicates. Error bars indicate s.e.m. Note difference in y-axis scales between panels C and D.

Fig 4: Engineered CHO-K1 N1D1 + Rfng cells show ligand-dependent cis-activation.(A) Schematic of actual and potential cis- and trans-interaction modes in the Notch pathway. (B) Schematic of the N1D1 + Rfng cell line. CHO-K1 cells were engineered to express a chimeric receptor combining the Notch1 extracellular domain (‘N1ECD’, green) with the Gal4 transcription factor (orange) in place of the endogenous intracellular domain. When activated, released Gal4 activates a stably integrated fluorescent H2B-Citrine reporter gene (yellow) through UAS sites (brown) on the promoter. Cells also contain a stably integrated construct expressing Dll1 (red) with a co-translational (2A, white) H2B-mCherry readout (‘mCH’, red), from a 4-epiTc-inducible promoter. Cells also constitutively express Rfng (purple) and H2B-Cerulean (‘H2B-Cer’, blue). (C) (Left) Schematic of cis-activation assay conditions. A minority of N1D1 + Rfng (blue nuclei) cells were mixed with an excess of wild-type CHO-K1 cells (white nuclei). The typical distance between N1D1 + Rfng cells is ?x. (Right) Filmstrip showing activation (Citrine fluorescence, green) of an isolated N1D1 + Rfng cell using time-lapse microscopy. Constitutive cerulean fluorescence (blue) in the same cell nucleus is also shown (see Video 1 for additional examples). (D) Peak Notch activation rate in isolated N1D1 + Rfng cells (y-axis) versus distance to each of its closest neighboring N1D1 + Rfng cell (x-axis) at the point of maximum activity. One cell width is indicated by gray shaded area. Solid blue line indicates linear fit, whose flat slope suggests a cell-autonomous, distance-independent process. (E) Box plots showing the distribution of peak Notch activation rates in isolated N1D1 + Rfng cells prior to the first cell division in the cis-activation assay, for three different median Dll1 induction levels (indicated by numbers below bars; see Figure 1—figure supplement 2A for corresponding distributions).