Extended Data Fig. 9: Nfkbia basal expression predicts LPS-induced response in RAW cells, relative to Fig. 5.

(a) (Left panels) Overlaid mCherry intensity profiles of individual mock-treated (N = 23), LPS-treated (N = 122), or Live-seq-sampled and then LPS-treated (N = 77) cells. (Right panel) Area Under the Curve (AUC) values for all profiles shown in the left panels. P values were determined by a two-sided Wilcoxon rank-sum test. (b) Relative to Fig. 5a, linear relationship between the time post-LPS treatment (within a 3–7.5h window) and the Tnf-mCherry fluorescence intensity (log transformed) in one cell (see Supplementary Fig. 7 for all other cells). (c) Similar to Fig. 5b, a linear regression model was used to predict the intercept (basal Tnf-mCherry intensity) calculated from data shown in (b and Supplementary Fig. 7) based on ground-state Live-seq-recorded expression data. The Tnf gene is highlighted. (d) Basal Gsn expression anticorrelates with the rate of Tnf-mCherry fluorescence intensity increase. R² and FDR values are listed. (e) Similar to Fig. 5d, but the expression correlation between Nfkbia and Tnf is shown separately for RAW_Mock and RAW_LPS cells. The R² and P (F test) values of the linear regression model are shown. (f) Nfkbia is among the most variably expressed LPS-NF-kB pathway genes in primary macrophage cell populations. The expression and the variance of all expressed genes are shown, with genes of the KEGG NF-κB signaling pathway (downstream of the TLR4 receptor) highlighted. (g) Representative Tnf-mCherry and Nfkbia-BFP profiles from a single cell. Similar to endogenous Nfkbia, an in-house engineered Nfkbia-BFP reporter is induced by LPS treatment, synchronously with the Tnf-mCherry reporter, acting as a proxy for Nfkbia expression. LPS was applied at time 0 h (vertical dashed line). (h) Validation of Nfkbia expression as a predictor of a macrophage’s response to LPS (Methods). The basal Nfkbia level, here reflected by Nfkbia-BFP reporter intensity, negatively correlates with the rate of LPS-induced Tnf-mCherry intensity increase. Independent, biological replicate relative to Fig. 5e. R² = 0.10, P = 0.005, F-test; Pearson’s r = −0.34, P = 0.005. The error band (d, e and h) represents the SD.