Figure 9.
Spatiotemporal dynamics of CIP4 and ARHGAP17. (A) Representative TIRF micrographs of a SUM159 cell forming a single invadopodia after PDBu treatment expressing TKS5-mTagBFP2 as a marker for invadopodia, CIP4-GFP (expressed in CIP4 KD cells), and dTomato-wGBD as a marker for Cdc42 activity (scale bar, 0.5 μm). (B) Quantification of TKS5-mTagBFP2, CIP4-GFP (expressed in CIP4 KD cells), and dTomato-wGBD intensity at individual invadopodia. Intensity and time were rescaled between values of 0 and 1 for each invadopodia to allow for comparison. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (C) Cross-correlation analysis of TKS5-mTagBFP2, CIP4-GFP (expressed in CIP4 KD cells), and dTomato-wGBD intensity. Higher values denote positive correlation and lag denotes the time shift effect on correlation between signals. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (D) Invadopodia assay of SUM159 cells after 30 min PDBu treatment in which CIP4 and ARHGAP17 were either silenced and/or overexpressed in all possible combinations as indicated in the graph. 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. (E) Representative TIRF micrographs of ARHGAP17 KO SUM159 cells transfected with the indicated constructs and treated with PDBu for 30 min. Cells were fixed and stained for F-actin and stained for myc to detect myc-CIP4. Dashed lines mark the edge of the cell (scale bar, 10 μm). (F) Radial intensity analysis of F-actin, ARHGAP17-GFP, and myc-CIP4 in invadopodia. Lines are expressed as mean ± SEM of three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (G) Van Steensil’s cross-correlation function of ARHGAP17-GFP colocalization with myc-CIP4 or the background signal of the anti-myc antibody at invadopodia clusters in SUM159 cells treated with PDBu for 30 min. Shift denotes the x-dimension movement of one channel in relation to the other. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia clusters per condition per experiment were measured. (H) Representative TIRF micrographs of ARHGAP17 KO SUM159 cells forming single invadopodia after PDBu treatment expressing TKS5-mScarlet as a marker for invadopodia and either WT, S702E, or S702A ARHGAP17-GFP (expressed in ARHGAP17 KO background; scale bar, 0.5 μm). (I) Radial intensity profiles of individual invadopodia from live cells expressing TKS5-mScarlet as a marker for invadopodia and either WT, S702E, or S702A ARHGAP17-GFP (expressed in ARHGAP17 KO background) plotted over time. Time scales between invadopodia are rescaled between 0 and 1 to allow for alignment of intensity values. Results are expressed as the mean of at least eight representative invadopodia per condition. (J) Quantification of invadopodia lifetime in WT, S702E, or S702A ARHGAP17-GFP rescue SUM159 cells. Results are expressed as mean ± SEM of at least 200 invadopodia per condition. Significance was tested with a two-tailed t test: ***, P < 0.001.

Spatiotemporal dynamics of CIP4 and ARHGAP17. (A) Representative TIRF micrographs of a SUM159 cell forming a single invadopodia after PDBu treatment expressing TKS5-mTagBFP2 as a marker for invadopodia, CIP4-GFP (expressed in CIP4 KD cells), and dTomato-wGBD as a marker for Cdc42 activity (scale bar, 0.5 μm). (B) Quantification of TKS5-mTagBFP2, CIP4-GFP (expressed in CIP4 KD cells), and dTomato-wGBD intensity at individual invadopodia. Intensity and time were rescaled between values of 0 and 1 for each invadopodia to allow for comparison. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (C) Cross-correlation analysis of TKS5-mTagBFP2, CIP4-GFP (expressed in CIP4 KD cells), and dTomato-wGBD intensity. Higher values denote positive correlation and lag denotes the time shift effect on correlation between signals. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (D) Invadopodia assay of SUM159 cells after 30 min PDBu treatment in which CIP4 and ARHGAP17 were either silenced and/or overexpressed in all possible combinations as indicated in the graph. 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. (E) Representative TIRF micrographs of ARHGAP17 KO SUM159 cells transfected with the indicated constructs and treated with PDBu for 30 min. Cells were fixed and stained for F-actin and stained for myc to detect myc-CIP4. Dashed lines mark the edge of the cell (scale bar, 10 μm). (F) Radial intensity analysis of F-actin, ARHGAP17-GFP, and myc-CIP4 in invadopodia. Lines are expressed as mean ± SEM of three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (G) Van Steensil’s cross-correlation function of ARHGAP17-GFP colocalization with myc-CIP4 or the background signal of the anti-myc antibody at invadopodia clusters in SUM159 cells treated with PDBu for 30 min. Shift denotes the x-dimension movement of one channel in relation to the other. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia clusters per condition per experiment were measured. (H) Representative TIRF micrographs of ARHGAP17 KO SUM159 cells forming single invadopodia after PDBu treatment expressing TKS5-mScarlet as a marker for invadopodia and either WT, S702E, or S702A ARHGAP17-GFP (expressed in ARHGAP17 KO background; scale bar, 0.5 μm). (I) Radial intensity profiles of individual invadopodia from live cells expressing TKS5-mScarlet as a marker for invadopodia and either WT, S702E, or S702A ARHGAP17-GFP (expressed in ARHGAP17 KO background) plotted over time. Time scales between invadopodia are rescaled between 0 and 1 to allow for alignment of intensity values. Results are expressed as the mean of at least eight representative invadopodia per condition. (J) Quantification of invadopodia lifetime in WT, S702E, or S702A ARHGAP17-GFP rescue SUM159 cells. Results are expressed as mean ± SEM of at least 200 invadopodia per condition. Significance was tested with a two-tailed t test: ***, P < 0.001.

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