Fig 1: Oscillatory NFκB in response to PAMPs is a hallmark of feedforward TNF(A) Activity onset times in single-cell NFκB responses to 100 nM CpG, grouped by dynamic subtypes of the response (persistent, oscillatory, or transient).(B) Early-phase TNF secretion dynamics from macrophages stimulated with 100 nM CpG, as measured by ELISA.(C) Top: median surface TNFR1 expression over time in BMDMs exposed to 1 ng/mL TNF or 100 nM CpG, monitored by flow cytometry. Bottom: median surface TNFR1 expression over time in wild-type or Tnf−/− BMDMs in response to 100 nM CpG (scaled to receptor levels before treatment). Error bars show standard deviations across three independently performed experiments, and double asterisks indicate a p value <0.001 using a Student’s t test comparing wild-type and Tnf−/− levels at a particular timepoint.(D) Single-cell heatmaps of NFκB activation in RelAV/V BMDMs in response to 100 nM CpG, with or without feedforward TNF signaling blocked using saturating amounts (5 mg/mL) of soluble TNFR2 co-injected with treatment.(E) Proportions of NFκB dynamic subtypes (off, transient, oscillatory, or persistent) as quantified from the data in (D).(F) Schematic depicting two cells. One cell (left) responds to CpG by activating NFκB and producing TNF that may act upon it in an autocrine manner. Another cell (right) does not respond to CpG (possibly because of low TLR9 expression), but responds to paracrine TNF and hence produces oscillatory NFκB activity.