NF-κB Dynamics Discriminate between TNF Doses in Single Cells Qiuhong Zhang, Sanjana Gupta, David L. Schipper, Gabriel J. Kowalczyk, Allison E. Mancini, James R. Faeder, Robin E.C. Lee  Cell Systems  Volume 5, Issue 6, Pages 638-645.e5 (December 2017) DOI: 10.1016/j.cels.2017.10.011 Copyright © 2017 The Authors Terms and Conditions

Cell Systems 2017 5, 638-645.e5DOI: (10.1016/j.cels.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Population-Level Data Cannot Distinguish between Switch-like and Graded Response Mechanisms (A) Schematic for (M1) a switch-like mechanism for activation where a cytokine dose increases the probability of an all-or-nothing response in each cell; (M2) a graded mechanism for single-cell activation in which each cell's response is graded in proportion with increasing cytokine dose; and (M3) a combined threshold with graded mechanism where single-cell responses are graded in proportion with cytokine dose only if the dose is greater than the cell's threshold for activation. (B) Simulated responses of single cells modeled with extrinsic noise (described in Figure S1). Each cell is initialized in a unique “cell state” that approximates its responsiveness to cytokine. Responses to four doses are compared in each model plotted as a raw response (left), or a normalized response that divides each cell's response by its cell state (middle). The fraction of non-responder cells is quantified for each dose (right). (C) Three mechanistically distinct models are indiscernible when using the average of single-cell responses to approximate a population-level measurement. See also Figure S1. Cell Systems 2017 5, 638-645.e5DOI: (10.1016/j.cels.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Heterogeneity of Responses to TNF between Cell Lines and Single Cells (A) Time courses for average nuclear RelA from fixed cells are shown for a panel of human cell lines exposed to indicated concentrations of TNF continuously or as a single 1-min pulse (solid orange curve). On average, 11,374 single cells were measured across the time points for each cytokine condition. Light colored lines indicate the SD of approximately 1,274 cells measured at each time point. Time points for fixation included 0, 10, 30, 60, 90, 120, 180, 240, and 360 min following exposure to TNF. (B) Descriptors used to quantify the response of cells to a cytokine. Fi, Fmax, and Ff, respectively, describe the initial, maximal, and final amount of nuclear RelA fluorescence. AUC, the area under the curve for the cytokine response. ΔAdapt quantifies the deviation from a perfect adaptive response. Ratein and Rateout quantify the maximal rate of nuclear entry and exit, respectively, for average of fixed-cell data. (C) Heatmaps for each descriptors quantified in a panel of cell lines exposed to indicated cytokine conditions. Formulae used to calculate ΔAdapt and fold change (Fold) are shown. See also Figure S2. (D) Time-lapse images of FP-RelA stably expressed in KYM1 cells exposed to a single 1-min pulse of 1 ng/mL TNF. Scale bar, 10 μm. See also Movie S1. (E) Time courses of nuclear FP-RelA density measured in single cells treated with a 1-min pulse of 1 ng/mL TNF. See also Figure S2 and Table S1. Cell Systems 2017 5, 638-645.e5DOI: (10.1016/j.cels.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Information Transmission Capacity of the TNF-NF-κB Pathway (A) Density plots of single-cell FP-RelA time courses for responses to TNF with indicated concentration and duration. Median of single-cell responses for each condition is shown in blue. Inset numbers indicate the total number of single-cell time courses collected (black), the number of cells with a significant amount of FP-RelA translocation (red or pink), and the fraction of non-responders (NR) for each condition. (B) Channel capacity values calculated for each dataset: (dark blue) Raw and Fold datasets where each single-cell time course is represented in a.u. or fold change (Figure S3A); (light blue) NRR, datasets where time courses for non-responder cells are removed, the “Fold cont.” dataset only includes conditions from the Fold-NRR with continuous exposure to TNF, bottom row of (A); (red) average of 20 subsample control datasets where the same number of cell trajectories are removed from the Fold dataset as in the NRR, but cells were either “Randomly Selected” (Fold RS) or “Responding cells were targeted for Removal” (Fold RR) (see the STAR Methods). For all datasets, conditions with fewer than 100 responder cells, pink numbers in (A) were removed from channel capacity calculations; ∗p < 10−12, t test. (C) Channel capacity values for scalar descriptors of FP-RelA dynamics (∗p ≪ 10−13, t test). Error bars represent SD. See also Figure S3. Cell Systems 2017 5, 638-645.e5DOI: (10.1016/j.cels.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Repeat TNF Stimulation Reveals a Graded Mechanism of Dose Discrimination in Single Cells (A) Time-lapse images of FP-RelA in live cells stimulated with a 1-min reference pulse of 0.2 ng/mL TNF, followed by a 1-min pulse with 5× the reference dose (1 ng/mL TNF). Same cells are marked. Scale bar, 10 μm. See also Movie S2. (B) Representative single-cell time courses for the cells numbered 1–4 in (A). Inset shows schematic of AUC calculations for single-cell responses to the first (AUC1) and second (AUC2) TNF pulse. (C) Density plots of single-cell FP-RelA time courses for cells exposed to a reference TNF pulse followed by a range of increasing test doses. Median of single-cell responses is shown in blue and inset numbers indicate the number of single-cell time courses collected in each condition. (D) Scatterplots showing AUC2/AUC1 stratified along AUC1 across the range of test conditions. Colored bar along top depicts bins of single cells based on AUC1 into an approximately equal number of cells per condition. (E) Average response of single cells (AUC2) to increasing test pulse concentrations (0, 0.2, 1, 10, or 100 ng/mL TNF) for each bin in (D); inset describes Hill coefficients and goodness of fit for logistic regression. See also, Figure S4 and Table S2. Cell Systems 2017 5, 638-645.e5DOI: (10.1016/j.cels.2017.10.011) Copyright © 2017 The Authors Terms and Conditions