Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels Unimodal distribution of protein concentrations in artificial.

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Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels Unimodal distribution of protein concentrations in artificial cell populations. Distributions of basal concentrations for TGFβR1, SMAD2, and SMAD4 are shown for a population of artificial cells assembled according to the observed proportion of signaling classes at 100 pM TGFβ using calibrated noise levels. Silhouette plots of artificial cells sorted according to TGFβ concentration (left panel) or mapped to experimentally observed signaling classes (right panel). Plots provide a graphical representation of how well the nuc/cyt SMAD2 ratios of each simulated cell corresponds to trajectories of other simulated cells in its own group. Positive silhouette scores indicate that SMAD2 responses are more similar to the own group, while negative scores signify that the corresponding trajectory is closer to any of the other groups. In general, signaling classes provide better separation than sorting according to stimulus levels. Transition between signaling classes depending on feedback strength. The response of a reassembled population of artificial cells to 5 and 25 pM TGFβ was simulated with reduced feedback expression as indicated and mapped to previously observed signaling classes (see Appendix II.H). Black lines and their thickness indicate the direction and extent of transitions between signaling classes. Transitions with a probability below 1% are excluded for better visualization. Robustness of model predictions concerning SMAD7 knock‐out effect on distribution of signaling classes at 100 pM (top) and 5 pM (bottom) TGFβ, respectively. The simulated fraction of cells in each signaling class in wt and 30% feedback depleted cells is shown for 30 independent fits with similar goodness of fit obtained from local multistart optimization (see Appendix IV.D). The corresponding best‐fit results are shown in Fig A and G. Artificial cell populations were generated by adding the same protein concentration noise as in the best‐fit model (Fig C). White lines indicate median; boxes include data between the 25th and 75th percentiles; whiskers extend to maximum values within 1.5× the interquartile range; crosses represent outliers. Sequence of SMAD7 knock‐out alleles. The indicated sequence (red) in the second exon of the SMAD7 gene was targeted by Cas9 in combination with a specific sgRNA. This led to deletions of 22 nt and 7 nt in the targeted alleles, causing frameshifts and non‐sense mutations in the SMAD7 gene. Expression of SMAD7 mRNA in parental and knock‐out cells. Basal SMAD7 mRNA levels were determined by qPCR in the indicated cell lines. SMAD7 mRNA containing a prematures stop codon is degraded in knock‐out cells due to non‐sense mediated decay. β‐Actin was used as an internal control. Error bars indicate standard deviation of technical triplicates. Time‐resolved analysis of SMAD2 nuclear/cytoplasmic translocation in the absence of SMAD7. Median nuc/cyt SMAD2 ratio of cells stimulated with varying concentrations of TGFβ over 24 h are shown for parental (left) and SMAD7 knock‐out cells. See Appendix Table S1 for number of cells analyzed. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. Mol Syst Biol, Volume: 14, Issue: 1, First published: 25 January 2018, DOI: (10.15252/msb.20177733)