Myosin and actin polymerization inhibitors were added to the cultures before IR treatments. In Drosophila cells, treatment conditions were: 2 mM or 10 mM BDM (Sigma, Ref. 63 (link)) for 5 min; 200 or 400 μM MyoVin-1 (Calbiochem, Ref. 64 (link)) for 2 h; 100 μM or 400 μM CK666 (Sigma, Ref. 65 (link)) for 60 min; 4 μM or 8 μM Latrunculin B (Enzo Life Sciences) for 20 min. Stocks of MyoVin-1 (100 mM), CK666 (100 mg/ml) and Latrunculin B (10 mM) were prepared in DMSO and those of BDM were diluted in water. For mouse cells, treatment conditions were: 7.5 mM BDM for 20 min; 200 μM MyoVin-1 for 60 min; 400 μM CK666 for 60 min; 8 μM Latrunculin B for 20 min. All stocks were prepared in DMSO. For controls, DMSO was added to the media to yield final concentrations equivalent to samples treated with chemicals. For the ‘release’ time points of Extended Data Figs 1f–g and 6h, cells were washed 3× after incubation with each chemical, and incubated at 27 °C for 1 h before IR. Cells were fixed 60 min after IR. Notably, the doses of LatB treatments used affect nuclear actin polymerization in addition to cytoplasmic actin66 (link).