Figure 4.

Differential temporal activation of ERK1/2 in TNF-induced necroptosis compared to hFasL-induced apoptosis

(A–E) biosensing experiments: L929 expressing EKAR4.0 were treated with DMSO as a control (A), hFasL (B), TNF (C), and Nec1S orSCH772984 (added 1h in pretreatment) + TNF (D and E, respectively). Samples were time lapse-imaged by FRET microscopy for 30 min before treatment and 14.5 h after stimulation at the rate of 1 acquisition every 4 min. The FRET/CFP ratio for at least 100 cells in each cellular context (survival, apoptosis, and necroptosis) is displayed using a kymographic representation in which each row represents one cell. Cells were ordered by the duration of cell life, from top to bottom. Data shown are from at least two independent replicates. The time scale is at the bottom of each kymograph, and the color-coded fire LUT denotes FRET ratio changes and, therefore, reflects the changes in ERK1/2 activation over time. Black color indicates dead cells or missing points during the automatic analysis. Associated time-lapse images for each condition at different time points (0, 2, 4, and 6h) are displayed. The color-coded LUT reports on both FRET ratio changes and fluorescence intensity (i1 = 50; i2 = 1300). Scale bar: 20 μm. EKAR4.0 signal in each condition was then averaged. The mean (dark curve) and the SEM (grey errors bars) are plotted as a function of time. The number of cells analyzed is reported on each graph (n).