Figure 5.
GFPpkp4 and GFPp120 promote formation of distinct types of AJs. (a) Western blot of WT A431, δCat-KO derivatives, and their GFPpkp4- or GFPp120-expressing variants probed for E-cadherin, p120, pkp4, ARVCF, and β-tubulin. Molecular weight markers are as in Fig. 4 a. Both GFPp120 and GFPpkp4 increase E-cadherin levels. Also note that GFPpkp4 is strongly overexpressed yet remains below GFPp120, whose expression approximately matches endogenous p120 in WT A431 cells. (b and c) Maximum-intensity x–y projections of δCat-KO cells expressing GFPpkp4 (b) and GFPp120 (c) stained for GFP (green) and vinculin or PLEKHA5 (red). Low-magnification panels show merged images only. Bar, 20 μm. Enlarged regions (marked by dashed boxes) are shown below in individual channels. Bar, 15 μm. Arrows indicate several sparse PLEKHA5-positive AJs in GFPp120-expressing cells. (d) Quantification of GFP/PLEKHA5 or GFP/vinculin heterochromatic pixels (representing sums of lateral and apical/basal AJs, respectively) expressed relative to total GFP signal (n = 6 from three independent images). Note, GFPp120 and GFPpkp4 exert opposite effects on lateral versus apical/lateral AJ abundance. Statistical analysis: two-tailed Student’s t test: ****, P <0.0001. The means ± SD are indicated by bars. (e and f) Time-lapse imaging of GFPpkp4 (e) and GFPp120 (f) cells acquired at 30-s intervals; 0- and 60-s frames are shown. Corresponding enlarged contact regions (dashed white boxes) are shown at the bottom (see also Videos 4 and 5). Apical (ap) and basal (bl) contact edges are indicated. Bars: 20 μm (top), 10 μm (bottom). (g) Representative trajectories of AJs (left) and schematic summaries of their net displacements (right) presented as in Fig. 3. (h) FRAP analysis of lateral AJs in the cell lines shown in a (n = 15; mean ± SD). Source data are available for this figure: SourceData F5.

GFPpkp4 and GFPp120 promote formation of distinct types of AJs. (a) Western blot of WT A431, δCat-KO derivatives, and their GFPpkp4- or GFPp120-expressing variants probed for E-cadherin, p120, pkp4, ARVCF, and β-tubulin. Molecular weight markers are as in Fig. 4 a. Both GFPp120 and GFPpkp4 increase E-cadherin levels. Also note that GFPpkp4 is strongly overexpressed yet remains below GFPp120, whose expression approximately matches endogenous p120 in WT A431 cells. (b and c) Maximum-intensity x–y projections of δCat-KO cells expressing GFPpkp4 (b) and GFPp120 (c) stained for GFP (green) and vinculin or PLEKHA5 (red). Low-magnification panels show merged images only. Bar, 20 μm. Enlarged regions (marked by dashed boxes) are shown below in individual channels. Bar, 15 μm. Arrows indicate several sparse PLEKHA5-positive AJs in GFPp120-expressing cells. (d) Quantification of GFP/PLEKHA5 or GFP/vinculin heterochromatic pixels (representing sums of lateral and apical/basal AJs, respectively) expressed relative to total GFP signal (n = 6 from three independent images). Note, GFPp120 and GFPpkp4 exert opposite effects on lateral versus apical/lateral AJ abundance. Statistical analysis: two-tailed Student’s t test: ****, P <0.0001. The means ± SD are indicated by bars. (e and f) Time-lapse imaging of GFPpkp4 (e) and GFPp120 (f) cells acquired at 30-s intervals; 0- and 60-s frames are shown. Corresponding enlarged contact regions (dashed white boxes) are shown at the bottom (see also Videos 4 and 5). Apical (ap) and basal (bl) contact edges are indicated. Bars: 20 μm (top), 10 μm (bottom). (g) Representative trajectories of AJs (left) and schematic summaries of their net displacements (right) presented as in Fig. 3. (h) FRAP analysis of lateral AJs in the cell lines shown in a (n = 15; mean ± SD). Source data are available for this figure: SourceData F5.

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