Loss of vimentin increases nuclear damage but does not alter lamin expression levels. (a–c) Immunofluorescence images showing lamin A and lamin B1/2 on the top and bottom of membranes with 3 µm pores (a); on the membrane bottom, vim^−/− mEFs exhibit nuclear abnormalities such as large nuclear blebs (b), and some blebs exhibit dilated lamin A networks (c). (d) Migration through 3-µm pores results in an increased percentage of nuclei with blebs in vim^+/+ and vim^−/− mEFs, but vim^−/− mEFs have a significantly higher percentage of migration-induced blebs (n = 70–115 per condition from n = 2 independent experiments). Data are presented as means ± standard error, as noted in the statistical analysis section. (e and f) Loss of vimentin increases the number of postmigration DNA double-strand break foci, as determined by immunofluorescence with anti-γH2AX in nuclei after mEFs have migrated to the bottom of membranes with 3 µm pores (n = 70–115 per condition from n = 2 independent experiments). (g) Foci appear throughout the nucleus and in protruding blebs. Vim^+/+ and vim^−/− mEFs express equivalent levels of lamin A, B1/2, and C, as shown by quantitative immunoblotting. Scale bars: a, 10 µm; b, 2 µm; c, 10 µm, 2 µm (magnification); f, 10 µm. NS, non significant.