Fig 2: Trans-activation properties depend on ligand and receptor identity, and are modulated by Lfng.(A) Mean trans-activation signaling strength for different ligand-receptor-Lfng combinations. In each case, expression of the mCitrine reporter was normalized by the cotranslational Notch reporter (mTurq2) fluorescence. These values were averaged across all single cells in each sample, then background subtracted and further normalized by the strongest signaling activity measured for each receiver clone in the experiment. Normalized signaling activities for sub-saturated data points (Methods) were further normalized to the mean expression of the sender population (Figure 1C) in each coculture (Methods), and values are relative to the overall maximum. Data are from five different Notch1 receiver clones and three different Notch2 clones, with at least three biological replicates per clone (Supplementary file 3). Cis-ligand was suppressed with high 4-epi-Tc concentrations in receivers with integrated ligands. The significance of pairwise differences in normalized signaling activity was evaluated through permutation testing (see Methods). Except where labeled with ‘n.s.’, all signaling strengths within each subplot are significantly different (p-val<0.05) from all other receptor-ligand combinations within the same Lfng or dLfng condition. See Figure 2—figure supplement 1B for more granular analysis of p-values. Colors indicate the identity of the trans-ligand expressed by cocultured sender cells. Receptor identity is indicated below plot and by marker type (Notch1=circles, Notch2=triangles). Here and in subsequent panels, error bars denote bootstrapped 95% confidence intervals (Methods), in this case sampled from the number of bioreplicates given in the legend—n1 (for Notch1) or n2 (for Notch2). (B) Effects of Lfng on Notch1 (left) and Notch2 (right) signaling. Non-ligand-normalized signaling activities were re-plotted in dLfng (x-axis) vs. Lfng (y-axis) conditions. Saturated data points, defined here as those with normalized signaling activity over 0.75 in both dLfng and Lfng conditions, were excluded. Colored lines are least-squares linear fits. Black dashed line indicates no effect of Lfng expression. (C) Plotting the mean slope in (B) from bootstrap analysis reveals the effects of Lfng on trans-signaling for the indicated ligand-receptor combinations (Methods). Asterisks denote the p-value of the test statistic from a one-sided Wilcoxon signed-rank test (*p-val<0.05; **p-val<0.01; ***p-val<0.001), reflecting whether the slope is greater or less than 1. Receiver identity is indicated by x-axis labels and marker type. Ligand identity is indicated by color and the following abbreviations, which are used here and in subsequent figures: D1=Dll1, D4=Dll4, J1=Jag1, J2=Jag2. (D) Trans-activation dose-response curves for CHO-K1 Notch1 or Notch2 receivers, expressing endogenous Fringes activated by a cocultured majority of the Tet-OFF inducible sender cell lines shown in Figure 1—figure supplement 3A. Colors indicate ligand identity as in (C). X-axis values are the mean of the mCherry fluorescence in senders used for each coculture sample data point. Y-axis signaling activity values are the mean of the distribution in mCitrine (reporter activity, A.U.) divided by mTurq2 (cotranslational receptor expression, A.U.). Solid lines are fits of the increasing phase of each dataset to activating Hill functions (Methods). Dotted gray horizontal lines represent half-maximal signaling activity for each receptor across all ligand inputs. (E) Mean saturating signaling activities for indicated ligand-receptor combination, estimated by bootstrapping the Hill fits in (D), and normalized by the response to Jag2 for the same receptor. Although the decreasing phases of the Dll1 curves were excluded, saturating activity estimates may be influenced by incomplete saturation and biphasic behavior. (F) Mean logarithmic sensitivities of signaling response to ligand expression from bootstrapped linear regressions to log-log transformed, sub-saturating data points (see also Figure 2—figure supplement 3). (G) Mean Hill coefficients (n) estimated from bootstrap analysis of apparently saturating ligand-receptor responses in (D). (H) Relative signaling strength (ligand potency) was computed by estimating the ligand expression level sufficient to reach threshold activity level (where Hill fits crossed dotted lines in 2D) for each ligand-receptor combination, inverting it, and further normalizing all values by the value obtained for Jag2-Notch2. Mean values and 95% confidence intervals were computed from bootstrapped Hill function fitting (Methods). (I) Comparison of signaling strengths for Notch2 vs. Notch1 receivers in the dLfng, Lfng, and endogenous Fringe (enFng) backgrounds. dLfng and Lfng data are identical to values plotted in (A), and enFng values are identical to values plotted in (H), but scaled such that Dll4-Notch1 signaling strengths match in dLfng and enFng (based on Figure 2—figure supplement 4B).

Fig 3: Cis-inhibition strengths depend on the identities of the receptor, the cis-ligand, and the trans-ligand.(A) Results of cis-modulation assays for CHO-K1 Notch1 (top row) and Notch2 (bottom row) receivers with endogenous Fringes, coexpressing cis-ligands at different levels corresponding to a range of 4-epi-Tc concentrations (Supplementary file 2). Columns are cis-ligands, and colors indicate the ligand expressed by the cocultured sender cells (Figure 6—figure supplement 1A and B; Supplementary file 3). X- and y-axis values are flow cytometry fluorescence values averaged across all cells in a given mCherry bin (Figure 1B). The x-axis represents cis-ligand expression (mCherry, A.U.) relative to cotranslational receptor expression (mTurq2, A.U.); see also Methods. Y-axis values are Notch signaling activity (reporter activity [mCitrine, A.U.] divided by cotranslational receptor expression [mTurq2, A.U.]). For each bioreplicate curve, signaling activity was normalized to maximum trans-signaling, at the y-value with minimum cis-ligand (Figure 6—figure supplement 1C). Cis-inhibition of Jag1-Notch1 signaling could not be analyzed because trans-activation was too weak. For Notch2-Delta receivers, lines connect means of three biological replicates in each mCherry bin along the x-axis. For other receivers, curves are fits of three biological replicates to a repressive Hill function with maximum y=1 (Methods). (B) Mean cis-inhibition strengths based on bootstrap analysis of repressive Hill fits in (A). Cis-inhibition strength is defined as the inverse of the fit EC50 parameter, relative to the minimum value across all combinations. (C) Comparison of cis-inhibition strengths (y-axis, mean values and confidence intervals from (B) with Dll1 senders rescaled such that the maximum value over all ligand-receptor combinations equals 1) vs. trans-activation strengths with endogenous Fringes (x-axis, mean values and confidence intervals from Figure 2H). Cis-inhibition strength was set to zero for Delta-Notch2 combinations. (D) Cis-inhibition efficiencies and confidence intervals from (B) adjusted for the strength of trans-signaling induced by the indicated sender cells (Figure 6—figure supplement 1C), and normalized such that the maximum cis-inhibition strength is equal to 1 for each cis-ligand-receptor combination. Error bars reflect uncertainty on cis-inhibition efficiency only (trans-activation error bars were not propagated). (E) Hill coefficients (n) computed from fits of data in (A) to repressive Hill functions. Hill coefficients could not be computed for Notch1-Dll4 receivers with Dll4 or Jag2 senders because of the modest cis-activation observed at intermediate cis-ligand levels for those combinations. Values are means and 95% confidence intervals from bootstrap analysis.