Automated, repeated TNFα stimulations can artificially induce p65 oscillations and alter cell behavior. (A-B) Repeated addition and removal of TNFα (frequency: 20/70 minutes +/- TNFα) by a custom pipetting robot can force p65 to oscillate in GMP^GM. Dynamics (for 12 hours) and fate quantification (for 48 hours, cells cultured −TNFα) as depicted in Figure 5A (n = 190 cells, N = 3 biological replicates). (A) Left: signaling dynamics for different stimulation conditions. Gray/black lines: individual cells/means. Right: response dynamics frequencies. (B) Selected dynamics features for different stimulation conditions. Pulsed TNFα stimulation selectively forces OSC dynamics without changing the AUC (total activity over time) of p65 responses. (C) Selected fate readouts for different stimulation conditions. Forced p65 oscillations lead to smaller cells with a trend (P > .05) toward increased CD115 expression, and longer average division time, same as naturally OSC (GMP^M) cells (see also Figure 5D).