Fig. 6

MCAM regulates sc polarity and transmigration. a Polarity assays of SkMel2 cells expressing GFP-labelled wild-type (wt) ezrin or ezrin TA or TD mutants (n = 6, paired t-test). Representative images are shown in Supplementary Fig. 8c. b Representative images of SkMel2 cells in suspension expressing MCAM-GFP (MCAM) or MCAMΔKKGK-GFP (ΔKKGK) and ezrin-mCherry (ezrin) stained with DAPI. Scale bars: 10 µm. c Polarity assays of SkMel2 cells expressing ezrin-GFP (ctrl.) together with MCAM-mCherry (MCAM) or MCAMΔKKGK-mCherry (ΔKKGK) (n = 6, paired t-tests). MCAM expression was evaluated by mCherry fluorescence. d Polarity assays and representative images of SkMel2 cells expressing ezrin-mCherry (ctrl.) with GFP-ICAM (ICAM) or YFP-L1-CAM (L1-CAM) (n = 6, paired t-test). Scale bars: 10 µm. e Polarity assays of SkMel2 cells expressing ezrin-GFP (ctrl.) with non-targeting (n.t.) or MCAM-targeting (MCAM kd) siRNAs (n = 6, paired t-test). MCAM expression is shown in Supplementary Fig. 8d. f Adhesion of SkMel2 cells transfected with non-targeting (n.t.) or MCAM-targeting (MCAM kd) siRNAs was measured simultaneously in flow chambers. Cells were either unlabelled (white) or labelled with CellTracker green (green). The number of attached cells was quantified after 30 min. In all, 70–290 cells were analysed per measurement (n = 3, one-sample t-test compared to 50). g Transmigration assays of SkMel2 cells expressing no construct (ctrl.), mCherry (Cherry), MCAM-mCherry (MCAM) or MCAMΔKKGK-mCherry (ΔKKGK) through HUVEC (left) or through membrane only (right) showing the number of transmigrated cells after 24 h normalised to ctrl. (n = 4 (left), n = 5 (right), paired t-tests). h Transmigration assays of SkMel2 cells expressing no siRNA (ctrl.), n.t. or MCAM-targeting (MCAM kd) siRNAs as described in g. (n = 9 (left), n = 5 (right), paired t-tests). i Polarity assays of SkMel2 cells expressing MCAM-mCherry (ctrl.) with ezrin-GFP showing the fraction of MCAM polarised cells (n = 6, mean ± SD, paired t-test). Ezrin expression was evaluated by GFP fluorescence. All data represented as mean ± SD