VIFs hinder migration through small pores but limit nuclear deformation. Cells are seeded on Transwell membranes and allowed to migrate through pores in the membrane (see schematic). (a) Loss of vimentin increases migration through membranes of varying pore sizes, and normal cell motility is restored upon vimentin reexpression (n = 50–150 per condition from n = 2 independent experiments). Data are presented as means ± standard error, as noted in the statistical analysis section. (b) Images of vim^+/+ cells fixed and stained for vimentin and the nucleus (Hoechst) on the top and bottom of the membrane. Scale bar is 20 µm. (c) The membranes select for smaller nuclei, as shown by the decrease in nuclear area for cells on top and bottom of the membrane (n = 70–100 per condition from n = 2 independent experiments). (d) Pore migration increases nuclear aspect ratio (n = 70–115 per condition from n = 2 independent experiments). The lower and upper parts of the box represent the 25th and 75th percentiles. The ends of the notch indicate 1.57 times the interquartile range divided by the square root of the number of samples. The whiskers indicate the range of the circular variance up to 1.5 times the interquartile range away from the quartiles. In cells lacking vimentin, nuclei are stretched to a greater extent. Immunostaining shows nuclear morphologies on the bottom of the membrane in vim^+/+ and vim^−/− mEFs. The sample image of the vim^−/− nucleus is an example of the exceptionally large deformations seen in cells lacking vimentin. Scale bar is 10 µm. ns, non significant.