ARHGAP17 targets invadopodia through both the N-BAR and Proline-rich domains. (A) Domain structure of ARHGAP17 and the ARHGAP17 mutants used in this study. (B) Representative TIRF micrographs of individual invadopodia in SUM159 cells expressing ARHGAP17-GFP domain deletion mutants in ARHGAP17 KO cells. Cells were treated with PDBu for 30 min and stained for F-actin as a marker for invadopodia (scale bar, 0.5 μm). (C) Radial intensity analysis of F-actin and ARHGAP17-GFP mutants in invadopodia. Lines are expressed as mean ± SEM of four independent experiments in which at least 26 invadopodia per condition per experiment were measured. (D and E) Quantification of ARHGAP17 distance from the center of invadopodia based on peak intensity and quantification of normalized intensity of ARHGAP17 at invadopodia. Results are expressed as mean ± SEM of four independent experiments in which at least 26 invadopodia per condition per experiment were measured. Large circles represent biological replicates, small circles represent technical replicates. Significance was tested with a two-tailed t test: *, P < 0.05; ***, P < 0.001. (F) Percentage of cells with invadopodia in CTRL, ARHGAP17 KO, and Rescue with ARHGAP17 domain deletion mutants after 30 min PDBu treatment. Results are expressed as mean ± SEM of three independent experiments in which at least 200 cells per condition per experiment were counted. Statistical analysis is a one-way ANOVA with a Tukey’s post hoc test. Different letters indicate significant differences between conditions as defined by P < 0.05.