Figure S3.
CDC42EP5 modulates cytoskeletal features. (A) Image showing acetylated tubulin (red), GFP (green) and DAPI (blue) in 690.cl2 expressing GFP-Cdc42ep5 on glass. Right panels show single greyscale channel magnifications of the indicated area. Scale bars, 12.5 µm. (B) Images show F-actin (magenta), GFP (green), pS19-MLC2 (cyan), and DAPI (gray) signal of 690.cl2 cells stably expressing GFP-Cdc42ep5 on glass. Top panels show merged channel images of a protrusion (left) and perinuclear areas (basal, middle panel; apical, right panel). Bottom right inserts show whole-cell images. Lower panels show individual grayscale channels for the indicated regions and signals. Scale bars, 10 µm. (C) Images show F-actin (magenta) and DAPI (blue) staining of mouse embryonic fibroblasts (MEFs; top panels) and human melanoma cells (WM266.4, lower panels) on glass after transfection with control (siCtr) and CDC42EP5 (siEP5) siRNA. Grayscale magnifications of F-actin staining in indicated perinuclear areas are shown. Scale bars, 12.5 µm. Violin plot shows perinuclear F-actin intensity. n, single regions from individual cells; Mann–Whitney test: #, P < 0.0001. (D) Images of 690.cl2 cells after transfection with control (siCtr) and Cdc42ep5 (siEp5#1&3) siRNA on glass. Images show F-actin (magenta), Zyxin (green), and DAPI (blue) staining. Right panels show greyscale Zyxin channel magnifications. Scale bars, 12.5 µm. Graph shows percentage of cells with Zyxin-positive FAs for cells seeded on glass or on fibronectin. Bars indicate mean ± SEM (n = 4 experiments; unpaired t tests: #, P < 0.0001). (E) Plots show single-cell trajectories of 690.cl2 cells after transfection with control (siCtr) and two individual Cdc42EP5 (siEP5#1 and siEP5#3) siRNAs on 2D surfaces. Violin plot shows speed of individual cells. n, individual cells; Kruskal–Wallis and Dunn's tests: *, P < 0.05; #, P < 0.0001. (F) Western blots show pY438-Src and tubulin levels in 690.cl2 cells after transfection with control (siCtr) and siRNAs targeting individual Borg genes (siEp1–5). Graph shows the quantification of normalized pY418-Src levels. Bars indicate mean ± SEM (n = 5 experiments; one-way paired ANOVA, Dunnett’s test: †, not significant; ***, P < 0.001). AU, arbitrary units.

CDC42EP5 modulates cytoskeletal features. (A) Image showing acetylated tubulin (red), GFP (green) and DAPI (blue) in 690.cl2 expressing GFP-Cdc42ep5 on glass. Right panels show single greyscale channel magnifications of the indicated area. Scale bars, 12.5 µm. (B) Images show F-actin (magenta), GFP (green), pS19-MLC2 (cyan), and DAPI (gray) signal of 690.cl2 cells stably expressing GFP-Cdc42ep5 on glass. Top panels show merged channel images of a protrusion (left) and perinuclear areas (basal, middle panel; apical, right panel). Bottom right inserts show whole-cell images. Lower panels show individual grayscale channels for the indicated regions and signals. Scale bars, 10 µm. (C) Images show F-actin (magenta) and DAPI (blue) staining of mouse embryonic fibroblasts (MEFs; top panels) and human melanoma cells (WM266.4, lower panels) on glass after transfection with control (siCtr) and CDC42EP5 (siEP5) siRNA. Grayscale magnifications of F-actin staining in indicated perinuclear areas are shown. Scale bars, 12.5 µm. Violin plot shows perinuclear F-actin intensity. n, single regions from individual cells; Mann–Whitney test: #, P < 0.0001. (D) Images of 690.cl2 cells after transfection with control (siCtr) and Cdc42ep5 (siEp5#1&3) siRNA on glass. Images show F-actin (magenta), Zyxin (green), and DAPI (blue) staining. Right panels show greyscale Zyxin channel magnifications. Scale bars, 12.5 µm. Graph shows percentage of cells with Zyxin-positive FAs for cells seeded on glass or on fibronectin. Bars indicate mean ± SEM (n = 4 experiments; unpaired t tests: #, P < 0.0001). (E) Plots show single-cell trajectories of 690.cl2 cells after transfection with control (siCtr) and two individual Cdc42EP5 (siEP5#1 and siEP5#3) siRNAs on 2D surfaces. Violin plot shows speed of individual cells. n, individual cells; Kruskal–Wallis and Dunn's tests: *, P < 0.05; #, P < 0.0001. (F) Western blots show pY438-Src and tubulin levels in 690.cl2 cells after transfection with control (siCtr) and siRNAs targeting individual Borg genes (siEp1–5). Graph shows the quantification of normalized pY418-Src levels. Bars indicate mean ± SEM (n = 5 experiments; one-way paired ANOVA, Dunnett’s test: †, not significant; ***, P < 0.001). AU, arbitrary units.

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