Cytoplasmic BAF predominantly localizes to nuclear ruptures, and its behavior is primarily driven by association with DNA. (A) The nucleoplasmic/cytoplasmic GFP-BAF intensity was measured in live NIH3T3 cells following nuclear rupture with a blunted microcapillary. (B) The ratio of cytoplasmic/nucleoplasmic mCherry-NLS intensity was measured to monitor the repair of the ruptures. The shaded areas represent SEM (n = 5 cells). (C and D) Sequential images of NIH3T3 cells expressing GFP-BAF for which the GFP-BAF signal was photobleached in either the cytoplasm (n = 4 cells; C) or nucleoplasm (n = 4 cells; D), followed by laser-induced NE rupture (purple arrowheads). Yellow arrowheads indicate accumulation of GFP-BAF from each representative compartment at the site of nuclear rupture. (E) NIH3T3 cells expressing GFP-tagged BAF mutants, reduced DNA/histone-binding affinity (K6A), reduced LEM-domain protein-binding capability (L58R), double mutant (K6A, L58R), phosphomimetic (MEEEQ), or nonphosphorylatable (MAAAQ) underwent nuclear bleach then were ruptured using laser ablation. Migration of mutated BAF into the nucleus was monitored and compared with the WT BAF nuclear migration. (F) The average intensity of BAF was measured in regions of interest (ROIs) located in the nucleoplasm either distal (green circle) or proximal (orange circle) to the rupture site. The proximal-to-distal average intensity ratio for each cell was calculated for the first 3 min after nuclear rupture. The graph represents mean values ± SEM (n = 5 cells for each; **, P < 0.0001 from WT by a mixed-effects model with Tukey’s post hoc comparison test). Bars: 10 µm.