Figure 2.
JAM-A regulates CIL. (A) 1D kinematic CIL assays. Ctrl KD MCF7 or JAM-A KD MCF7 cells (EGFP-positive) were co-cultured with WT MCF7 (LifeAct-mCherry-positive) on linear micropatterns (width: 5 µm) and observed by live microscopy for 15 h. Cartoons: Types of CIL behavior after cell–cell collision: opposite migration (Type −2), anergy (Type −1), cell–cell contact formation (Type 0), and continuous migration (Type +1). Bottom panels: Still images of movies representative of different CIL behavior. (B) Quantification of CIL types after cell collisions of scrambled shRNA-expressing MCF7 cells (Ctrl KD) and JAM-A shRNA-expressing MCF7 cells (JAM-A KD). Number of collisions: n = 113 for Ctrl KD–WT (four independent experiments), n = 56 for JAM-A KD–WT (four independent experiments), n = 88 for JAM-A KD—JAM-A KD (3 independent experiments). (C) Early cell–cell contact formation in JAM-A-depleted MCF7 cells. MCF7 cells with doxycycline-inducible KD of JAM-A (pEmU6proT plasmid; shRNA expression off (Ctrl KD), shRNA expression on (JAM-A KD) were fixed during early cell–cell contact formation. Arrowheads indicate sites of E-cadherin-positive puncta. Scale bars: 5 µm. (D) MDA-MB-231 cell collision assays of scrambled shRNA-expressing cells (Ctrl KD) or JAM-A shRNA-expressing cells (JAM-A KD) with wild-type cells (WT). Number of collisions: n = 73 for Ctrl KD–WT, n = 50 for JAM-A KD—WT (three independent experiments). (E) MCF7 cell collision assays of scrambled shRNA-expressing cells (Ctrl KD) or JAM-A shRNA-expressing cells (JAM-A KD) on laminin (LN) (n = 90 for Ctrl KD–WT, n = 157 for JAM-A KD–WT, four independent experiments) and fibronectin (FN) (n = 118 for Ctrl KD–WT, n = 143 for JAM-A KD—WT, four independent experiments). Symbols above horizontal bars represent comparisons with VN-cultured MCF7 cells shown in Fig. 2 B. (F) Mixing assays of MCF7 cells. Ctrl KD: pEmU6proT plasmid; − dox: shRNA expression off. JAM-A KD: pEmU6proT plasmid; + dox: shRNA expression on. Cells co-express either LifeAct-EGFP or LifeAct-mCherry as indicated. Left: Fluorescence images of cell collectives (top), and binary images highlighting areas of overlap between colliding leader cells (bottom). Right: Quantification of the areas of overlap. Data is derived from four independent experiments. Scale bars: 10 µm. 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 < 0.001; ****, P < 0.0001.

JAM-A regulates CIL. (A) 1D kinematic CIL assays. Ctrl KD MCF7 or JAM-A KD MCF7 cells (EGFP-positive) were co-cultured with WT MCF7 (LifeAct-mCherry-positive) on linear micropatterns (width: 5 µm) and observed by live microscopy for 15 h. Cartoons: Types of CIL behavior after cell–cell collision: opposite migration (Type −2), anergy (Type −1), cell–cell contact formation (Type 0), and continuous migration (Type +1). Bottom panels: Still images of movies representative of different CIL behavior. (B) Quantification of CIL types after cell collisions of scrambled shRNA-expressing MCF7 cells (Ctrl KD) and JAM-A shRNA-expressing MCF7 cells (JAM-A KD). Number of collisions: n = 113 for Ctrl KD–WT (four independent experiments), n = 56 for JAM-A KD–WT (four independent experiments), n = 88 for JAM-A KD—JAM-A KD (3 independent experiments). (C) Early cell–cell contact formation in JAM-A-depleted MCF7 cells. MCF7 cells with doxycycline-inducible KD of JAM-A (pEmU6proT plasmid; shRNA expression off (Ctrl KD), shRNA expression on (JAM-A KD) were fixed during early cell–cell contact formation. Arrowheads indicate sites of E-cadherin-positive puncta. Scale bars: 5 µm. (D) MDA-MB-231 cell collision assays of scrambled shRNA-expressing cells (Ctrl KD) or JAM-A shRNA-expressing cells (JAM-A KD) with wild-type cells (WT). Number of collisions: n = 73 for Ctrl KD–WT, n = 50 for JAM-A KD—WT (three independent experiments). (E) MCF7 cell collision assays of scrambled shRNA-expressing cells (Ctrl KD) or JAM-A shRNA-expressing cells (JAM-A KD) on laminin (LN) (n = 90 for Ctrl KD–WT, n = 157 for JAM-A KD–WT, four independent experiments) and fibronectin (FN) (n = 118 for Ctrl KD–WT, n = 143 for JAM-A KD—WT, four independent experiments). Symbols above horizontal bars represent comparisons with VN-cultured MCF7 cells shown in Fig. 2 B. (F) Mixing assays of MCF7 cells. Ctrl KD: pEmU6proT plasmid; − dox: shRNA expression off. JAM-A KD: pEmU6proT plasmid; + dox: shRNA expression on. Cells co-express either LifeAct-EGFP or LifeAct-mCherry as indicated. Left: Fluorescence images of cell collectives (top), and binary images highlighting areas of overlap between colliding leader cells (bottom). Right: Quantification of the areas of overlap. Data is derived from four independent experiments. Scale bars: 10 µm. 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 < 0.001; ****, P < 0.0001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal