Confinement promotes nuclear blebbing and rupture via a RhoA/ROCK/myosin-II/SUN1/2–dependent pathway. (a) Representative images of the nucleus in 2D and confinement as visualized from cells fixed and stained with Hoechst. White arrow indicates nuclear bleb. Scale bars, 5 µm. (b) Percentage of HT-1080 cells exhibiting nuclear blebbing in 2D versus confinement (n ≥ 3 independent experiments with a minimum of 30 cells per experiment). (c) Time required for the first incident of nuclear blebbing versus nuclear rupture as observed from HT-1080 cells expressing NLS-MBP-GFP-NES(Rev) and H2B or NUP50-mCherry (n = 3 independent experiments with a minimum of five cells per experiment). (d) Image sequence of an HT-1080 cell experiencing transient nuclear rupture in confined channels visualized by NLS-MBP-GFP-NES(Rev) mislocalization in the nucleus and NUP50-mCherry mislocalization in the cytoplasm. Scale bar, 10 µm. (e) Percentage of HT-1080 cells experiencing nuclear rupture in 2D and confinement, as quantified from mislocalization of NLS-MBP-GFP-NES(Rev) from the cytoplasm to the nucleus (n = 4 independent experiments with a minimum of eight cells per experiment). (f) Percentage of control, 10 µM Y27632-treated, and constitutively active RhoA (Q63L) HT-1080 cells displaying nuclear blebbing, as observed from cells fixed and stained with Hoechst (n ≥ 3 independent experiments with a minimum of 15 cells per experiment). (g) Percentage of scramble control, MIIA-knockdown, MIIB-knockdown, and dual MIIA/B-knockdown HT-1080 cells displaying nuclear blebbing, as observed from cells fixed and stained with Hoechst (n ≥ 3 independent experiments with a minimum of 15 cells per experiment). (h) Percentage of scramble control, SUN1-knockdown, and/or SUN2-knockdown HT-1080 cells displaying nuclear blebbing (n ≥ 3 independent experiments with a minimum of 15 cells per experiment). Values represent mean± SEM. **, P < 0.01 relative to 2D; #, P < 0.05; ##, P < 0.01 relative to confined control or scramble control (SC).