Fig 2: 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 trans-activation 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.

Fig 3: Similar ligand-receptor signaling features occur in CHO-K1 and C2C12-Nkd cells.(A) Normalized Notch signaling strength in C2C12-Nkd Notch1 (black) or Notch2 (green) receivers cultured with Jag1 (y-axis) vs. Dll1 (x-axis) senders in a trans-activation assay. Receivers were treated with negative control (unfilled markers) or mouse Rfng (filled markers) siRNAs prior to the assay. X and y-axis values are mean signaling activity (reporter activity, mCitrine, divided by cotranslational receptor expression, mTurq2), background subtracted and normalized to the average signal in Dll1 coculture with negative control siRNA treatment for each receiver. Solid lines are the least-squares best fit through all points for a given receiver (pooling control and Rfng siRNA-treated samples, see Methods). Both slopes were significantly greater or less than 1 according to one-sided Wilcoxon signed-rank tests (p-val<0.05). The black dashed line is y=x. (B) The cell density used in the C2C12-Nkd cis-activation assay prevents intercellular signaling. After siRNA treatment to knock down residual endogenous Notch components (Methods), the assay was performed similarly to the assay used for CHO-K1 cells (Figure 4A), except for use of a 12-well plate. Here, y-axis values are min-max normalized, mean Notch signaling activities. Black bars are the mean of three biological repeats. (C) Results of cis-activation (left column) and cis-modulation (right column) assays for C2C12-Nkd Notch1 (top row) and Notch2 (bottom row) receivers coexpressing a Notch ligand or control protein. Fluorescence values were averaged differently for Notch1 vs. Notch2 based on responses to the nerve growth factor receptor (NGFR) control (see Methods). For both receptors, signaling activity defined in (A) was min-max normalized using the maximal trans-signaling in receivers cultured with the high-Dll4 senders (Dll4-2H10) used in the cis-modulation assay (Supplementary file 3). Lines connect means of three biological replicates (individual data points) in each mCherry bin. The bottom right plot is a zoomed-in view of the data for Notch2 receivers cultured with high-Dll4 senders, showing Jag2 cis-inhibition of Notch2 activation by Dll4 senders. (D) Comparison of cis-ligand effects in the cis-modulation assay for CHO-K1 (‘CHO’) vs. C2C12-Nkd (‘C2C12’) cell types. Y-axis units are normalized signaling activities as defined in Figure 4B for CHO-K1 cells and in (C) for C2C12-Nkd cells. (Note, maximal trans-signaling activities used in normalization differed greatly for CHO-K1 and C2C12-Nkd, so responses should be compared qualitatively, but not quantitatively (Methods).) Here, y-values are signaling activities corresponding to a cis-ligand expression level where x-axis cotranslational H2B-mCherry fluorescence equals 3×104 A.U., calculated by fitting a line between the x-axis mCherry bins flanking x=3×104 A.U. Y-values are the mean, and error bars are 95% confidence intervals, from bootstrap analysis.

Fig 4: 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.

Fig 5: 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).