Figure 3.
JAM-A limits cell motility in single cells. (A) Positioning of cells in CIL type +1 events observed after collisions of JAM-A KD MCF7 cells (pLVTHM JAM-A shRNA) with MCF7 WT cells. Cells were cultured on VN-coated linear micropatterns (5 µm width). Number of collisions analyzed: n = 68 (six independent experiments). (B) Quantification of displacement (left panel) and migration velocity (right panel) of single cells cultured on VN-coated linear micropatterns (5 µm width). Analysis was performed using the TrackMate Plugin for ImageJ software. Number of cells analyzed: n = 67 for MCF7 WT cells, n = 64 for JAM-A KD cells (three independent experiments). (C) Quantification of mean velocity, directionality and motility coefficient of single cells cultured on 2D areas of VN-coated micropatterns. Number of cells analyzed: n = 105 for control MCF7 cells (JAM-A shRNA not induced, Ctrl MCF7), n = 112 for JAM-A KD cells (JAM-A shRNA induced, JAM-A KD MCF7; five independent experiments). (D) Quantification of mean velocity of single cells cultured on 2D areas of LN-coated and FN-coated micropatterns. Number of cells analyzed: LN: n = 59 for control MCF7 cells (JAM-A shRNA not induced, Ctrl MCF7), n = 74 for JAM-A KD cells (JAM-A shRNA induced, JAM-A KD MCF7; three independent experiments). FN: n = 67 for control MCF7 cells, n = 78 for JAM-A KD cells (three independent experiments). (E) Analysis of cellular stiffness by AFM. Data is expressed as Relative Young’s Modulus (left panel, n = 522 for ctrl cells, pLVTHM-EGFP control vector, n = 516 for JAM-A KD cells, pLVTHM JAM-A shRNA vector, three independent experiments), and as peak adhesion force (right panel, n = 571 for ctrl cells, n = 578 for JAM-A KD cells, three independent experiments). Statistical analysis was performed using Wilcoxon Signed Rank Test. ****, P < 0.0001. Unless stated otherwise, all statistical analyses were performed with unpaired Student’s t test. Data are presented as mean values ± SD. NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

JAM-A limits cell motility in single cells. (A) Positioning of cells in CIL type +1 events observed after collisions of JAM-A KD MCF7 cells (pLVTHM JAM-A shRNA) with MCF7 WT cells. Cells were cultured on VN-coated linear micropatterns (5 µm width). Number of collisions analyzed: n = 68 (six independent experiments). (B) Quantification of displacement (left panel) and migration velocity (right panel) of single cells cultured on VN-coated linear micropatterns (5 µm width). Analysis was performed using the TrackMate Plugin for ImageJ software. Number of cells analyzed: n = 67 for MCF7 WT cells, n = 64 for JAM-A KD cells (three independent experiments). (C) Quantification of mean velocity, directionality and motility coefficient of single cells cultured on 2D areas of VN-coated micropatterns. Number of cells analyzed: n = 105 for control MCF7 cells (JAM-A shRNA not induced, Ctrl MCF7), n = 112 for JAM-A KD cells (JAM-A shRNA induced, JAM-A KD MCF7; five independent experiments). (D) Quantification of mean velocity of single cells cultured on 2D areas of LN-coated and FN-coated micropatterns. Number of cells analyzed: LN: n = 59 for control MCF7 cells (JAM-A shRNA not induced, Ctrl MCF7), n = 74 for JAM-A KD cells (JAM-A shRNA induced, JAM-A KD MCF7; three independent experiments). FN: n = 67 for control MCF7 cells, n = 78 for JAM-A KD cells (three independent experiments). (E) Analysis of cellular stiffness by AFM. Data is expressed as Relative Young’s Modulus (left panel, n = 522 for ctrl cells, pLVTHM-EGFP control vector, n = 516 for JAM-A KD cells, pLVTHM JAM-A shRNA vector, three independent experiments), and as peak adhesion force (right panel, n = 571 for ctrl cells, n = 578 for JAM-A KD cells, three independent experiments). Statistical analysis was performed using Wilcoxon Signed Rank Test. ****, P < 0.0001. Unless stated otherwise, all statistical analyses were performed with unpaired Student’s t test. Data are presented as mean values ± SD. NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

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