CYRI-A and CYRI-B cooperatively regulate cell shape and migration. (A) Immunofluorescence images of control (pLKO), single KO, and DBKO of CYRIs in A-673 cells. Cells stained for F-actin with phalloidin. Orange arrowheads indicate C-shape cells. Scale bar = 50 µm. (B) Western blots showing the efficiency of single KO and DBKO of CYRIs. GAPDH as loading control. (C) Quantification of cell shape (left) and spread area (right) of A from at least 50 cells per experiment from three independent experiments. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ns, P > 0.05; **, P < 0.01; ****, P < 0.0001. (D and E) Migration analysis of CYRI CRISPR cells on a 2D fibronectin substrate (random migration) or in 3D CDM. Data from at least 30 cells per experiment from three independent experiments. Each experiment is color-coded. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ****, P < 0.0001. (F) Reexpression of CYRI-A in DBKO A-673 cells reduces their speed to the original values but does not affect the control pLKO cells. Data from at least 30 cells per experiment from three independent experiments. Each experiment is color-coded. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ns, P > 0.05; ****, P < 0.0001. (G and H) Wound healing assay comparing the control pLKO, single KO, and DBKO CYRI cells. Data from at least four independent experiments. Each experiment is color-coded. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. Orange dotted lines highlight the edge of the cell monolayer. (I) Proliferation assay using the Incucyte system of control pLKO, single KO, and DBKO CYRIs in A-673 cells. Data from at least three independent experiments. Mean ± SEM.