CDC42EP5 interacts with SEPT9 to promote actomyosin activity. (A) Western blot showing Sept9 and GFP levels in total lysates (input) and anti-GFP immunoprecipitates (IP:GFP) in 690.cl2 cells ectopically expressing GFP, GFP-Cdc42ep5^WT, and GFP-Cdc42ep5^GPS-AAA. (B) Western blot showing Cdc42ep5 and Gapdh expression in parental 690.cl2 cells (wild-type) and 690.cl2^KO cells expressing GFP, GFP-Cdc42ep5^WT, or GFP-Cdc42ep5^GPS-AAA. (C) Images show GFP (green), Sept9 (blue), and F-actin (magenta) in 690.cl2^KO cells expressing GFP, GFP-Cdc42ep5^WT, or GFP-Cdc42ep5^GPS-AAA on glass. Bottom panels are merged and single-channel magnifications of perinuclear areas. Scale bars, 20 µm. Violin plot in the right shows perinuclear Sept9 mean intensity in the indicated cells. n, individual cells; Kruskal–Wallis and Dunn's tests: †, not significant; #, P < 0.0001. (D) Graph shows the relative frequencies of roundness indexes in 690.cl2^KO cells expressing GFP, GFP-Cdc42ep5^WT or GFP-Cdc42ep5^GPS-AAA on collagen-rich matrices (additional representation of Fig. 6 C). (E) Western blot showing pS19-MLC2 and Sept2 expression in 690.cl2^KO cells expressing GFP, GFP-Cdc42ep5^WT, or GFP-Cdc42ep5^GPS-AAA. Graph shows the normalized pS19-MLC2 levels. Bars indicate mean ± SEM (n = 3 experiments; one-way ANOVA, Tukey’s test: †, not significant; *, P < 0.05; **, P < 0.01). (F) Graph shows the relative frequencies of roundness indexes in 690.cl2^KO cells expressing GFP or GFP-SEPT9_V1 on collagen-rich matrices (additional representation of Fig. 6 F). AU, arbitrary units; KO, knockout.