ROCK phosphorylates FHOD3 and promotes formation of actin spike protrusions. (A) A2780 cells stably expressing RFP or RFP-FHOD3/shFHOD3#1 were untreated or treated with cRDGfV (2.5 µM, 30 min) and ROCK inhibitor Gly-H1152 (100 nM, 30 min) or Rho inhibitor I (4 µg/ml, 4 h) as indicated. RFP trap was performed from cell lysates, and subjected to SDS-PAGE and Western blotting using RFP and pS/T antibodies. Fluorescence intensity of pS/T bands was measured using ImageJ, and expressed relative to untreated (n > 4 per condition). Error bars represent the SEM. (B) A2780 cells were seeded onto CDMs and treated with cRGDfV (2.5 µM) and ROCK inhibitor Gly-H1152 (100 nM) where indicated before time-lapse imaging. Asterisks indicate the position of individual cells at different time points. (C) Migration speed was measured for cells treated as in B; n = 150 cells per condition. (D and E) A2780 cells transiently transfected with Lifeact-mEGFP were seeded onto 3D CDM for 4 h, and cRGDfV (2.5 µM, 2 h) and Gly-H1152 were added as indicated. Z stacks were captured on a spinning disk confocal microscope every minute for ≥25 min. Z projections are shown. White arrows indicate lamellipodia-like actin veils; red arrows indicate actin spikes. Boxed regions are enlarged on the right. (E) Normalized protrusion size was measured for cells in D; n = 21–27 cells per condition. All data represent at least three independent experiments; statistical significance was evaluated using ANOVA/Tukey’s multiple comparison test. +, mean; n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.