N-cad endocytosis, recycling, and surface levels in leader and follower cells. (A) Schematic diagram of the N-cad antibody (Ab) internalization assay. Cells were labeled with N-cad ECD Ab in the cold, warmed for various times to allow internalization, fixed, and surface and internalized Ab detected with different fluorescent secondaries. (B) Immunofluorescence detection of internalized N-cad Ab with EEA1, Rab5, or Rab4 in leader cells after 40 min internalization. Arrowheads indicate colocalization. Scale bars, 10 or 2 μm (inset). (C) Object-based colocalization quantification of internalized N-cad Ab with EEA1, Rab5, Rab4, Rab11, LAMP1, or GM130. N = 5–10 spheroids, three experiments. (D) Surface N-cad Ab increases between 40 and 60 min; a.u., arbitrary units. Two-way ANOVA Šídák’s multiple comparisons test multiple comparisons test. *P < 0.05, ***P < 0.001. N = 3–4 spheroids, three experiments. (E and F) Schematic and representative images of N-cad recycling assay. Cells were incubated with N-cad ECD antibody in the cold and warmed for 40 min to allow internalization. N-cad Ab remaining on the surface was blocked in the cold with excess F(ab’)2. Cells were then warmed to allow recycling before fixation and detection of surface and internalized Ab with different fluorescent secondaries. Scale bars, 10 μm. (G and H) Schematic diagram and representative images showing photoconversion of histone H2B-Dendra2 in leader and follower cells. (I–K) Flow cytometry. (I) Definition of leader, follower and spheroid cells according to extent of photoconversion. (J) N-cad surface intensity histograms. (K) Normalized median N-cad fluorescent intensity. Paired t test. *P = 0.0374. N = 3 experiments. Error bars show mean ± SEM.
