Nesprin-2 knockdown recapitulates NMIIB shRNA phenotype. (A) Western blot of nesprin-1 or -2 expression levels. MDA-MB 231 cells were infected with lentivirus expressing shRNA sequences of either NT control, nesprin-1 shRNA targets, or nesprin-2 shRNA targets. To account for off-target effects, two separate constructs were used for nesprin-1 and -2. (B) Collagen invasion of nesprin-1 or -2 shRNA cells (n = 16 fields analyzed). (C) Nuclear translocation time of H2B-RFP–labeled nuclei through 5-µm-wide pores (shaded bars), compared with nonrestrictive control channels (open bars) of 3D migration devices (n = 30–50 nuclei). (D) Nuclear height of nesprin knockdown cells measured from x–z projections of DAPI-stained nuclei (n = 60–70 nuclei). Error bars represent SEM. **, P < 0.01; ***, P < 0.001.