MDA-MB-231 or U2OS cells stably expressing clover-ERK-KTR were seeded on fibronectin-coated (1 µg /ml) Ibidi 8-well slides (Ibidi) 1 day before imaging. Four hours before imaging, the medium was supplemented with 250 nM sir-DNA (Cytoskeleton) and 25 mM HEPES (Sigma). Cells were then imaged live (37 °C, 5% CO2) using a Nikon Eclipse Ti2-E microscope (Nikon) equipped with an sCMOS Orca Flash4.0 camera (Hamamatsu) and controlled by the NIS-Elements software (Nikon, v 5.11.01). MDA-MB-231 cells were imaged using a 20× Nikon CFI Plan Apo Lambda objective (NA 0.75), either 1 frame per minute for 2 hours or 1 frame every 5 minutes for 17 hours. In these experiments, a camera binning of 2 × 2 was used. This dataset consists of 20 videos.