Signaling properties of recombinant ligands
(A) Plated ligand assay records Notch receivers’ responses to plated recombinant human C-terminal Fc-tagged ligand extracellular domains (“ligand-ext-Fc”) (schematic). Blue in the cell nucleus represents H2B-mTurq2 fluorescence (readout of receptor expression), and the yellow construct represents the mCitrine reporter promoter. Line-dot-and-arrow icon refers to this assay.
(B) CHO-K1 Notch1 (left) and Notch2 (right) receivers expressing endogenous Fringes were cocultured on plated recombinant ligand-ext-Fc proteins at the concentrations indicated on the x-axis. Y-axis signaling activity values are the mean of mCitrine reporter distributions (reporter activity, A.U.) divided by mTurq2 (cotranslational receptor expression, A.U.). Solid lines are activating Hill function fits, with free Hill coefficient (n) and EC50 parameters. Saturating activities were fixed since the curves did not fully saturate in these ligand concentration regimes (Methods). Dotted gray horizontal lines represent the signaling thresholds defined in Figure 2D.
(C) Mean signaling strengths, based on bootstrap analysis of the responses in (D) (Methods). Each signaling strength is defined as the inverse of the ligand concentration sufficient to reach threshold activity level (dotted lines in (B)), normalized to show receptors’ relative activities. Colors and labels indicate ligand identity, as in Figure 2. X-axis labels are receivers; ‘N’ = ‘Notch.’ Here and in subsequent panels, error bars denote bootstrap 95% confidence intervals (Methods).
(D) Comparison of mean Notch2/Notch1 signaling strength ratios with canonical transactivation in sender-receiver cell cocultures (y-axis, values from Figure 2H) vs. the plated ligand assay (x-axis, values from (C)).
(E) Mean logarithmic sensitivities computed from the slope of linear regressions to 10,000 bootstrap replicates of log-log sub-saturating signaling activities vs. ligand concentrations in Figure 3—figure supplement 1.
(F) Soluble ligand binding assay (schematic, see also Methods), which enables the quantification of the strength of receptor binding to ligand-ext-Fc pre-clustered with secondary antibody. Blue in the cell nucleus represents H2B-mTurq2 fluorescence (readout of receptor expression). Star-dot-and-arrow icon refers to this specific assay.
(G) Scatterplot of averaged single-cell data from the soluble ligand binding assay with Lfng (y-axis) vs. dLfng (x-axis) expression (Methods). Fluorescence background, determined as ligand bound to parental reporter cells with no ectopic Notch receptors, was subtracted, and negative values were set to zero. Solid lines are least-squares best-fits, and the black dashed line is y=x.
(H) Mean fold difference in the amount of each ligand bound to Notch1 with Lfng vs. dLfng from slopes of linear regressions in (G), based on bootstrap analysis of n = 4 biological replicates per ligand. X-axis labels are recombinant ligands.
(I) Normalized Notch signaling strength in CHO-K1 Notch1 receivers expressing Lfng or dLfng, plated on Dll1-ext-Fc (yellow) or Jag1-ext-Fc (purple) in a plated ligand assay (Methods). X and y-axis values represent mean signaling activity (reporter activity, mCitrine, divided by cotranslational receptor expression, mTurq2), background subtracted and normalized to the maximum signaling activity—the average signal in the same receiver (+Lfng) plated on a high concentration of Dll4-ext-Fc. Solid lines are the least-squares best fits to six data points, representing three biological replicates for each of two plated ligand concentrations. Both slopes were significantly greater than or less than 1 according to a one-sided Wilcoxon signed-rank test (p-value = 0.015 for both lines). The black dashed line is y=x. Bracketed numbers are bootstrap 95% confidence intervals.
(J) Cell schematic depicting the effects of Lfng expression on Jag1 and Dll1 interactions with the Notch1 receptor. White triangles represent glycosylation modifications added by Lfng, and yellow saturation level in cell nuclei represents signaling activity.