NMIIB facilitates nuclear translocation through restrictive pores. (A) Schematic showing 3D chemotactic invasion chamber. Cells are seeded in the lower chamber in the diagram, and a chemoattractant gradient is established with 200 ng/ml EGF-containing media in the opposite chamber. Time-lapse images are then collected and the time of nuclear transit through restrictive pores is measured via kymograph analysis. White arrowhead indicates nonrestrictive control channel. White arrow indicates direction of cellular migration. (B) Representative time course images of an MDA-MB 231 cell expressing a Histone2B–CFP nuclear marker passing through a 5 × 5-µm² pore. The white dashed line in each frame represents the position used for kymograph generation. Bar, 50 µm. (C) Representative kymographs showing time courses of nuclei passing through multiple parallel channels (individual channels denoted by arrowheads) of an invasion chamber. MDA-MB 231 NT shRNA control cells (left) have a shorter nuclear retention in the pore, as compared with NMIIB knockdown cells (right). Vertical bar, 120 min. (D) Quantification of nuclear transit time. *, P < 0.05; ***, P < 0.0001 relative to NT control. Nuclear transit through 5-µm-wide restrictions in shaded bars and unrestricted nuclear transit in open bars. n = 60 to 100 nuclei analyzed per condition. (E) GFP-NMIIB transfection rescues efficient nuclear transit through 3D microfluidic invasion devices to control levels. Error bars indicate SEM; *, P < 0.05.