FHOD3 drives actin spike formation and migration in 3D matrix. (A) A2780 cells stably transfected with control or FHOD3 targeting shRNA constructs were lysed, then lysates were subjected to SDS-PAGE and Western blotting using antibodies specific for FHOD3 and α-tubulin, and analyzed using Odyssey. (B) A2780 cells stably expressing shFHOD3 #1, shFHOD3 #2, pLVTHM vector control, or RFP-FHOD3 rescue were seeded onto CDM and treated with cRGDfV (2.5 µM) where indicated before time-lapse imaging, and migration speed (B; n = 96–134 cells per condition) and pseudopod length (C; n = 100 cells per condition) were quantified. (D) Representative images of cells from B and C. Asterisks indicate the position of individual cells at different time points. (E) A2780 cells stably transfected as in B 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. Red arrowheads indicate actin spikes; blue arrowheads indicate blebs. White boxed regions are enlarged on the right. (F) Kymographs of yellow boxes in E. (G and H) Normalized protrusion size of cells (G; n = 23–28 cells per condition) and percentage of movie frames with bleb protrusions (H; n = 30 cells per condition). All data represent at least three independent experiments. Statistical significance was evaluated using ANOVA/Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.