SEPT9 is a crucial effector of CDC42EP5 function in melanoma. (A) Diagram showing the different domains in Cdc42ep5. Underneath are the amino acid sequences of the BD3 segment in wild-type and septin-binding–defective mutant (GPS-AAA). (B) Images show GFP (green), F-actin (magenta), pY118-Paxillin (cyan), and DAPI (gray) in 690.cl2^KO cells expressing GFP, GFP-Cdc42EP5^WT, or GFP-Cdc42EP5^GPS-AAA on glass. Single-channel magnifications of indicated areas are shown. Scale bars, 10 µm. Violin plots show quantification of perinuclear F-actin intensity (top) and FA size (bottom). n, independent regions in individual cells; one-way ANOVA Tukey’s test: †, not significant; *, P < 0.05; *, P < 0.05; #, P < 0.0001. (C) Images show GFP, F-actin and pS19-MLC2 in 690.cl2^KO cells expressing GFP, GFP-Cdc42EP5^WT, or GFP-Cdc42EP5^GPS-AAA on collagen-rich matrices. Scale bar, 75 µm. Bottom graphs show violin plots for roundness index (left) and pS19-MLC2 mean intensity (right) of individual cells. n, individual cells; Kruskal–Wallis and Dunn's tests: †, not significant; #, P < 0.0001. (D and E) Graphs show fold migration ability (D) and 3D collagen invasion (E) of 690.cl2^KO cells expressing GFP, GFP-Cdc42EP5^WT, or GFP-Cdc42EP5^GPS-AAA. Bars indicate mean ± SEM (n = 3 [D] and 6 [E]; one-way ANOVA Tukey’s test: †, not significant; *, P < 0.05; **, P < 0.01; #, P < 0.0001). (F) Images show F-actin (magenta), GFP (green), and pS19-MLC2 (cyan) in 690.cl2^KO cells expressing GFP or GFP-SEPT9_V1 on collagen-rich matrices. Individual channels are also shown. Scale bars, 75 µm. Violin plots show roundness index (left) and pS19-MLC2 mean intensity (right) of individual cells. n, individual cells; Mann–Whitney test. (G) Images showing GFP (green), F-actin (magenta), and pY118-Paxilin (cyan) in 690.cl2^KO cells expressing GFP or GFP-SEPT9_V1 on glass. Right panels show indicated magnifications. Scale bars, 10 µm. Violin plots show quantification of F-actin intensity in the perinuclear region (left) and of FA size (right). n, single regions from individual cells (left) and individual FAs (right); Mann–Whitney test. (H) Graph shows fold migration ability of 690.cl2^KO cells expressing GFP, GFP-SEPT9_V1, or GFP-Cdc42ep5^WT. Bars indicate mean ± SEM (n = 3 experiments; paired t test: *, P < 0.05). AU, arbitrary units. KO, knockout.