Extended Data Fig. 3: Cell elimination is independent of compressive stress or cell death.
From: Force transmission is a master regulator of mechanical cell competition
To quantify E-cad KO and WT cell extrusions and determine their position, we developed an automated workflow: (a) Exemplary raw data (merged brightfield and fluorescent signal) and segmentation of extrusions (white = WT, green = E-cad KO). The supervised learning algorithm ilastik was trained to detect and classify extrusions based on both signals. Details about the training and the accuracy can be found in the Methods section. (b) To determine accurate extrusion positions and avoid counting extrusions multiple times, the extrusions were tracked through time using TrackMate. (c) Average isotropic stress before E-cad KO and WT extrusions. T = 0 indicates detection, that is completion of the extrusion process. Stresses were measured within a square of size 60 µm around one extrusion event for different time points. n = 726 (E-cad KO) and n = 334 (WT) extrusions in n = 10 movies from N = 2 independent experiments. (d) Cell death is detected by the annexin V singal (magenta) in mixed culture. (e) Quantification of cell fate. For each extrusion, we calculated the time from cell elimination until detection of the annexin V signal. The plot shows the fraction annexin V-positive cells after the time of extrusion. n = 11327 extrusions from n = 17 movies and N = 4 independent experiments. (f) Representative images of mixed culture treated with a pan-caspase inhibitor (Z-VAD-FMK, 20 µM). Magenta line shows initial cluster boundaries. Right: Area quantification compared of the standard experimental condition. N = 1 caspase inhibitor experiment and n = 5 positions. Unless otherwise stated, data are presented as mean values +/− SD. Scale bars 100 µm (a, d, f); 25 µm (e).
