FHOD3 phosphorylation is required for actin spike protrusions and migration in 3D CDM. (A) A2780 cells stably expressing RFP or RFP-FHOD3 wt/3A/3D were treated with cRDGfV (2.5 mM, 30 min) and RFP trap as in Fig. 6 A. Fluorescence intensity of pS/T bands was measured using ImageJ, and expressed relative to untreated (n = 4 per condition). (B) A2780 cells were seeded onto CDM and treated with cRGDfV (2.5 µM) and ROCK inhibitor Gly-H1152 (100nM) where indicated before time-lapse imaging. Asterisks indicate the position of individual cells at different time points. (C) Migration speed was determined for cells treated as in B; n > 60 cells per condition. (D) A2780 cells stably expressing RFP/pLVTHM, RFP-FHOD3 3A/shFHOD3#1, or RFP-FHOD3 3D/shFHOD3#1 were transiently transfected with Lifeact-mTFP1, seeded onto CDM for 4 h, and treated with cRGDfV (2.5 µM) before spinning disk confocal imaging. Maximum z projections are shown. (E) Kymograph analysis of D, taken from the areas in yellow boxes. (F) Normalized protrusion size of cells in D. n = 22–27 cells per condition. All data represent at least three independent experiments. Statistical significance was evaluated using ANOVA/Tukey’s multiple comparison test. +, mean; *, P < 0.05; **, P < 0.01; ***, P < 0.001.