TEM of dye-labeled WT T cells was set up as in Figure 4. After 5 min of TEM under flow cells were fixed, permeabilized, and stained with DAPI and an anti-FMNL1 antibody (A) Representative maximum Z projections of a transmigrating T cell, defined as a cell with a portion of the cytoplasm underneath the endothelial monolayer. (B) Representative 3D side view reconstruction of the transmigrating T cell in A. Dashed line indicates position of the endothelial monolayer. (C) Representative linescan quantification of fluorescence intensities in a transmigrating T cell. Graph of the fluorescence intensities along the depicted line in each channel relative to the position within the cell. (D) FMNL1 is enriched behind the nucleus in transmigrating T cells. Percentage of transmigrating T cells with perinuclear enrichment behind the nucleus, partial enrichment of FMNL1 behind the nucleus, or no enrichment (see Methods for analysis). (E) FMNL1 deficiency impairs transmigration of the T cell nucleus across endothelial barriers. Position of T cell nuclei in fixed WT or FMNL1 KO T cells after 5 min of TEM under flow. Using spinning-disk confocal microscopy and DAPI staining, the nuclei were scored as being above the plane of the endothelium, in the process of transmigrating, or below. Data in D are the mean ± SEM from 3 independent experiments with >15 cells analyzed per experiment; data in E are the mean ± SEM from 4 independent experiments with >50 cells analyzed per experiment. Statistics in E were calculated using repeated-measures ANOVA with Sidak’s multiple comparisons test. n.s. = not significant, *=p < 0.05, **=p < 0.01.