Distinct N-cad localization and dynamics in leader and follower cells. (A) Localization of N-cad in PBT-05 cells migrating on laminin. Maximum intensity projections. Insets show enlarged orthogonal views of follower (r1) and leader (r2) cells. Arrowheads indicate cell-cell junctions and arrows indicate vesicles. Scale bars, 10 or 7 μm (inset). (B) Intracellular N-cad or transferrin receptor-positive vesicles per cell were counted in leader and follower cells. N = 3 experiments. Unpaired t test. ***P < 0.0001. ns, not significant. (C) Representative images showing colocalization between N-cad and endosomal markers. Arrowheads indicate N-cad-positive endocytic vesicles colocalized with Rab5 or Rab11. Scale bars, 10 or 2 μm (inset). (D) Mander’s colocalization coefficients. N = 7–12 cells, 2–3 spheroids for each marker protein analyzed. (E and F) N-cad antibody uptake assay. Surface N-cad was labeled with N-cad ECD antibodies on ice before warming for various times, fixing, and staining for surface and intracellular antibody as described in Fig. S3 A. (E) Representative images. Scale bars, 10 μm. (F) Quantification. a.u., arbitrary units. N = 7–24 cells, 2–4 spheroids, 2 experiments. Two-way ANOVA Tukey’s multiple comparisons test between leader and follower cells at the same time point. ****P < 0.0001. (G) Recycling of intracellular N-cad Abs to the surface in leader and follower cells, measured as described in Fig. S3 E. N = 4 experiments each representing the average of 10 cells in each of 3–5 spheroids. Paired t test. *P < 0.05. (H) Quantification of total N-cad fluorescence intensity in leader and follower cells. Migrating cells were fixed, permeabilized, and stained with N-cad Ab. Fluorescence intensity was integrated for multiple leader and follower cells across the z stack. Paired t test. ***P < 0.001. N = 8 spheroids, five experiments. Error bars show mean ± SEM.