Figure 5.

Differential patterns of EKAR4.0 pulse dynamics in TNF-induced necroptosis and hFasL-induced apoptosis compared to survival

(A) For each condition, similarly to the FRET/CFP ratio, a binary kymographic representation displays the corresponding EKAR4.0 pulse dynamics pattern.

(B) Automatic detection of ERK1/2 activity pulses was performed using a peak detection algorithm, and regions of increased ERK1/2 activity are identified by red dots on one representative FRET/CFP ratio profile for one cell and each condition, plotted against time. The green line corresponds to the time of stimulation. At the bottom of each graph, a corresponding heatmap of EKAR4.0 signal is shown with binary values (0–1 or dark blue to yellow, respectively), meaning a shift of ERK1/2 activity between the “OFF” state (EKAR4.0 FRET_OFF) to the “ON” state (EKAR4.0 FRET_ON). Black indicates dead cells.

(C) Time-resolved quantitative analysis of EKAR4.0 pulse dynamics based on the following parameters: pulse amplitude, pulse number per time fraction, pulse duration (min), and FRET_ON state of ERK1/2 (calculated for each cell, normalized to the cell life duration of each cell, and expressed as a percentage of time of the cell lifespan). These parameters were calculated 1h after the start of imaging with a 2h time interval for each cell and each condition. Data are presented as radar plots in which each spot corresponds to the mean of at least two independent experiments. Dark spots and lines indicate the maximum for each parameter, and the min and max values are reported as follows: pulses width (6–10 min); pulses number (4–6); pulses amplitude (1.1–1.4); FRET_ON (20–35%). Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. Significance between samples is indicated as follows: ^∗p< 0.05; ^∗∗p< 0.01; ^∗∗∗p< 0.001; ^∗∗∗∗p< 0.0001; ns, not significant.