Figure 6.
ARHGAP17 Cdc42-specific catalytic activity is required for its function at invadopodia. (A) Pulldown of endogenous ARHGAP17 in SUM159 cells using constitutively active RhoA (Q63L), Rac1 (Q61L), and Cdc42 (Q61L) tagged with GST or with GST alone. (B) Pulldown of active Cdc42 using the p21 binding domain (PBD) of p21 activated kinase 1 protein. Results are expressed as mean ± SEM of four independent experiments. Significance was tested with a two-tailed t test: ***, P < 0.001. (C and D) PBD pulldown of active Cdc42 in CTRL (C) and ARHGAP17 KD (D) SUM159 cells after treatment with PDBu at the indicated time points. Left panel shows a representative Western blot; right panel shows the quantification of three independent experiments. Results are expressed as mean ± SEM Significance was tested with a two-tailed t test: *, P < 0.05. (E) Representative TIRF micrographs of ARHGAP17 KO SUM159 cells forming single invadopodia after PDBu treatment stained for F-actin as a marker for invadopodia and expressing either WT or CD ARHGAP17-GFP (expressed in ARHGAP17 KO background; scale bar, 0.5 μm). (F) Radial intensity analysis of F-actin, WT ARHGAP17-GFP, and CD ARHGAP17-GFP in invadopodia. Lines are expressed as mean ± SEM of three independent experiments in which at least 40 invadopodia per condition per experiment were measured. (G and H) Quantification of ARHGAP17-GFP distance from the center of invadopodia based on peak intensity and normalized intensity of ARHGAP17 at invadopodia. Results are expressed as mean ± SEM of three independent experiments in which at least 40 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. (I) Percentage of cells with invadopodia in CTRL, ARHGAP17 KO, and Rescue with either WT or CD ARHGAP17-GFP 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. Significance was tested with a two-tailed t test: **, P < 0.01. For all blots, molecular weights are indicated in kD. Source data are available for this figure: SourceData F6.

ARHGAP17 Cdc42-specific catalytic activity is required for its function at invadopodia. (A) Pulldown of endogenous ARHGAP17 in SUM159 cells using constitutively active RhoA (Q63L), Rac1 (Q61L), and Cdc42 (Q61L) tagged with GST or with GST alone. (B) Pulldown of active Cdc42 using the p21 binding domain (PBD) of p21 activated kinase 1 protein. Results are expressed as mean ± SEM of four independent experiments. Significance was tested with a two-tailed t test: ***, P < 0.001. (C and D) PBD pulldown of active Cdc42 in CTRL (C) and ARHGAP17 KD (D) SUM159 cells after treatment with PDBu at the indicated time points. Left panel shows a representative Western blot; right panel shows the quantification of three independent experiments. Results are expressed as mean ± SEM Significance was tested with a two-tailed t test: *, P < 0.05. (E) Representative TIRF micrographs of ARHGAP17 KO SUM159 cells forming single invadopodia after PDBu treatment stained for F-actin as a marker for invadopodia and expressing either WT or CD ARHGAP17-GFP (expressed in ARHGAP17 KO background; scale bar, 0.5 μm). (F) Radial intensity analysis of F-actin, WT ARHGAP17-GFP, and CD ARHGAP17-GFP in invadopodia. Lines are expressed as mean ± SEM of three independent experiments in which at least 40 invadopodia per condition per experiment were measured. (G and H) Quantification of ARHGAP17-GFP distance from the center of invadopodia based on peak intensity and normalized intensity of ARHGAP17 at invadopodia. Results are expressed as mean ± SEM of three independent experiments in which at least 40 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. (I) Percentage of cells with invadopodia in CTRL, ARHGAP17 KO, and Rescue with either WT or CD ARHGAP17-GFP 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. Significance was tested with a two-tailed t test: **, P < 0.01. For all blots, molecular weights are indicated in kD. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal