Mechanical confinement of U2OS nuclei is sufficient for NE rupture. (A) Images of cell confinement device. (B) Schematic representation of confinement device as viewed from objective. Large squares represent wells, small squares represent areas under the confinement slide, and circles indicate areas where pillars contact well bottom (not to scale). Colors represent mean nucleus height from C. (C) Quantification of nucleus height of U2OS GFP-LmnB1 cells treated with indicated drugs and confined for at least 1 h before imaging. Boxes are quartiles and whiskers are 5th to 95th percentiles. n ≥ 60 nuclei; two-way analysis of variance, P < 0.001 for drug and confinement variables. **, P < 0.01; ***, P < 0.001 (multiple comparisons). (D) Images from 24-h time lapse of U2OS GFP-NLS shLmnB1 cells confined after treatment with indicated drugs. Arrows indicate sites of chromatin herniation. (E) Quantification of chromatin hernia formed during 24-h time-lapse imaging. Graph represents pooled data (columns) from two experiments (points, n > 150 cells). χ2 analysis P < 0.001, results of Fisher’s exact test between indicated pairs above the graph. ***, P < 0.001; ns, P > 0.05. (F) Quantification of NE rupture during 24-h time-lapse imaging. Graph represents pooled data (columns) from three experiments (points, n > 150 cells). χ2 analysis, P < 0.001, with results of Fisher’s exact test between indicated pairs shown above the graph. **, P < 0.01; ***, P < 0.001; ns, P > 0.05. (G) Model of interphase NE rupture in cultured cells. Bars, 10 µm.
