JAM-A prevents cell overgrowth and localizes to cellular protrusions. (A) Control MCF7 cells (pLVTHM, EGFP-positive) or JAM-A KD MCF7 cells (pLVTHM-JAM-A shRNA, EGFP-positive) were co-cultured with non-transfected MCF7 cells. Samples were stained for JAM-A. Right panels: Quantification of cellular overlap between Ctrl KD or JAM-A KD MCF7 cells (EGFP-positive) and WT MCF7 cells (LifeAct-mCherry-positive). Cell overlaps are depicted as pixels present in the area of overlap (Imaris software). Left diagram: JAM-A shRNA (number of analyzed contacts: n = 208 for Ctrl MCF7, n = 280 for JAM-A shRNA KD MCF7; three independent experiments). Right diagram: JAM-A siRNA pool (n = 166 for control siRNA pool, n = 196 for JAM-A siRNA pool; four independent experiments). (B) HEK293T cells transfected with WT JAM-A (Flag-JAM-A WT) or a dimerization mutant of JAM-A (Flag-JAM-A ER/KE) were co-cultured with untransfected HEK293T cells and stained with antibodies against Flag (green) and JAM-A (red). Collision events were divided into three categories: no overlap (coverage <10%), moderate overlap (coverage 10–50%), and strong overlap (coverage >50%). Number of events: n = 133 for Flag-JAM-A WT, n = 150 for Flag-JAM-A ER/KE. Data was obtained from three independent experiments. Frequency distributions were compared using a Chi-Square test. ****, P < 0.0001. Scale bars: 10 µm. (C) EGFP-JAM-A-expressing MCF7 cells were analyzed by live microscopy. Arrowheads indicate enrichment of JAM-A at protruding membranes. Scale bar: 10 µm. (D) Collectively migrating MCF7 cells were stained for αvβ5 integrin and JAM-A (top), or FAK and JAM-A (bottom). Arrowheads indicate co-localization of JAM-A with αvβ5 integrin (top) and FAK (bottom) at cell protrusions. Scale bars: 10 µm. (E) MCF7 cells were co-stained for αvβ5 integrin, JAM-A, and F-Actin (top), or for Src, JAM-A, and F-actin (bottom). Arrowheads indicate co-localization of JAM-A with αvβ5 integrin (top) and Src (bottom) at cell protrusions. Scale bars: 5 µm.