Cooverexpression of multiple DNA repair factors, soft ECM, and myosin II inhibition rescue rupture-induced DNA damage in low–lamin A cells. (A) siLMNA-U2OS cells were fixed after 24 h DNA repair factor transfection and immunostained for γH2AX. The nontransfected (NT) sample was also stained for KU80. Cytoplasmic mislocalization of the GFP or KU80 identifies ruptured nuclei. Bar graph: Cotransfection of DNA repair factors KU70, KU80, and BRCA1 (GFP-3) rescues excess DNA damage in ruptured nuclei, whereas ruptured nuclei with GFP-53BP1 (and NT) maintain excess DNA damage. Nonruptured cells always show a basal level of DNA damage. n = 30–100 cells per condition in three experiments. (B) Excess DNA damage and loss of repair factors in A549 shLMNA nuclei is suppressed by culturing on softer gels (3 or 0.3 kPa), whereas Ctl cells are unaffected. Bottom: Culturing A549 shLMNA cells on soft gels (0.3 kPa) leads to less stress fiber assembly in conjunction with rounder and smaller nuclei. n > 100 cells per condition in three experiments. (C) Nuclear envelope rupture and excess DNA damage (γH2AX foci counts and comet assay) in A549 shLMNA cells cultured on rigid plastic are reduced to Ctl levels after myosin II inhibition by blebbistatin, while Ctl cells are unaffected. n > 100 cells per condition in three experiments. *, P < 0.05. Bars, 10 µm. All data are present as mean ± SEM.