Fig. 9

Generic depolarisation decreases transmigration and seeding. a Polarity assays from 30 min (polarised) and 3 h (depolarised) time points from Fig. 2a (n = 6, paired t-test). b Transmigration assays of polarised (30 min) and depolarised (3 h) SkMel2 cells through HUVEC after 4, 7 or 24 h (n = 3, one-sample t-tests compared to 100). Further transmigration assays are shown in Supplementary Fig. 15f, g. c Adhesion assays of polarised (30 min) and depolarised (3 h) ezrin-GFP-expressing SkMel2 cells settling onto plastic. The area per cell was measured at the indicated times in >110 cells from at least 3 independent experiments (unpaired t-tests). d Individual (top) and averaged (bottom) adhesion kinetics of single polarised (n = 48, red) and unpolarised (n = 30, blue) ezrin-GFP-expressing SkMel2 cells within one, untreated population. Polarisation prior to adhesion was assessed at time point 0. The area per cell was measured at the indicated times (unpaired t-tests). e Adhesion of polarised and depolarised SkMel2 cells measured simultaneously in flow chambers after 30 min. Cells were unlabelled (white) or labelled with CellTracker green (green). In all, 70–380 cells were analysed per measurement (n = 4, one-sample t-test compared to 50). f, h Polarity assays of ezrin-GFP-expressing U87 (f) and SNU-1 (h) cells at 30 min (polarised) and 3 h (depolarised) time points (n = 6, paired t-test). g, i Transmigration assays of polarised (30 min) and depolarised (3 h) U87 cells (g) through HUVEC after 4, 7 or 24 h and of SNU-1 cells (i) after 5, 8 or 24 h through membrane (n = 3 (g), n = 4 (i), one-sample t-tests compared to 100). j 3D reconstruction of GFP stainings from 30 serial 2 µm sections (right) of mouse lungs 30 min after injection with polarised or depolarised ezrin-GFP-expressing SkMel2 cells (Supplementary Movies 17 and 18). Grid length: 100 µm. Scale bars: 100 µm, arrowheads: GFP-positive cells. k–m Quantification of GFP-positive SkMel2 (k), U87 (l) and SNU-1 (m) cells from histological slides of mouse lungs as shown in j and Supplementary Fig. 17c, f by GFP-positive area per haematoxylin-positive area (n = 10 (k), n = 7–8 (l), n = 13–15 (m), unpaired t-tests, normalised to the average of 'polarised' in m). Further quantifications are shown in Supplementary Fig. 18. All data are represented as mean ± SD