Overexpression of multiple DNA repair factors rescues DNA damage independently of rupture in constricted migration, but cell cycle suppression persists. (A) Constricted migration causes DNA repair proteins BRCA1 and 53BP1 to mislocalize from the nucleus (Nucl.) into the cytoplasm (Cyto.; more than five fields of view each; n > 3 experiments; *, P < 0.05). Bot., bottom; Int., intensity; anti-, antibody-stained. (B) As a nucleus squeezes out of a constriction, both endogenous KU80 (yellow) and overexpressed YFP-NLS (green) are mislocalized to cytoplasm, but in nuclei that have fully exited, KU80 remains cytoplasmic while YFP-NLS is mostly nuclear. Bar graphs: Overall nuclear/cytoplasm (Nucl./Cyto.) ratio is thus lower for KU80 than for YFP-NLS after 3-µm pore migration (more than five fields of view each; n > 3 experiments per condition; *, P < 0.05). (C) Co-overexpression of three DNA repair factors (KUsBR) partially rescues excess DNA damage after constricted migration despite nuclear rupture, whereas rescue is not achieved by overexpression of any single repair protein (200–500 cells per condition, n > 3 experiments per condition; *, P < 0.05.). n.s., not significant. (D) The 4N population (Pop.) of both control and KUsBR cells remain suppressed despite the partial rescue of migration-induced DNA damage (200–500 cells per condition, n > 3 experiments per condition; *, P < 0.05). All scale bars: 10 µm. (E) A critical amount of DNA damage is hypothesized to block cell cycle progression.