NMIIB localizes to the rear and perinuclear region of cells migrating through 3D environments. MDA-MB 231 cells were fixed and stained as they migrated through restriction points of the 3D migration devices. Dotted lines indicate positions of posts that create 5-µm pores. (A) Cells were stained for NMIIA and F-actin (phalloidin). Arrowhead marks leading edge where a high concentration of NMIIA can be observed. (B) Cells are stained for NMIIB and phalloidin. Arrowhead marks NMIIB localization in trailing edge. (C) Cells are stained for pMLC and phalloidin. Arrowheads mark perinuclear stress fibers containing pMLC. (D) Images from time-lapse showing MDA-MB 231 cell expressing nuclear marker H2B-RFP (red) and GFP-NMIIB (green) as it migrates through a 5-µm-wide restriction pore. Arrowheads indicate stress fibers above the nucleus containing GFP-NMIIB and arrows indicate posterior NMIIB accumulation. Bars, 10 µm.