Figure S6.
Cdc42 biosensor validation. (A) Representative micrographs of a SUM159 cell expressing the single-chain Cdc42 FRET biosensor and mCherry-cortactin as a marker for invadopodia after PDBu treatment (scale bar, 5 μm). The color calibration bar indicates the relative FRET ratio. White arrowheads indicate the invadopodia cluster; yellow arrowheads indicate the lamellipodia protrusion. (B) Quantification of FRET ratio and mCherry-cortactin intensity at individual invadopodia. Intensity and time have been rescaled between values of 0 and 1 for each invadopodia to allow for comparison. Lines are loess smoothing and 95% confidence from five invadopodia. (C) Representative micrographs highlighting Cdc42 FRET at invadopodia in a SUM159 cell as marked by mCherry-cortactin (scale bar, 5 μm). (D) Radial intensity analysis of FRET ratio and mCherry-cortactin intensity at invadopodia. Lines are expressed as mean ± SEM of 40 invadopodia. (E) Design of the Cdc42 activity biosensor used in this study, composed of the GTPase-binding domain of WASP (wGBD) tagged with dTomato. (F) Representative micrographs of SUM159 cells showing colocalization of the dTomato-wGBD sensor with different mTurquoise2-H2A-RhoGTPase-ΔCAAX constructs (RhoA-G14V, Rac1-G12V, or Cdc42-G12V; scale bar, 10 μm). (G) Quantification of the average intensity of dTomato-wGBD signal within the nucleus ratioed to the average intensity in the cytosol. Results are mean ± SD of at least 14 cells per condition from three independent experiments. Significance was tested with a two-tailed t test: ****, P < 0.0001. (H) Representative TIRF micrographs of lamellipodia protrusions from SUM159 cells expressing either dTomato as a control for cytoplasmic localization or dTomato-wGBD (scale bar, 5 μm). (I) Quantification of H in which a 10-pixel wide line was drawn perpendicular to the cell edge (yellow dashed line in H) and the intensity was measured for each frame. Distance 0 starts 10–15 μm within the cell and the protrusion can be visualized by a change in distance at intensity 0.25 over time. The increase in peak dTomato-wGBD intensity over time indicates an increase in Cdc42 activity localized to the leading edge. (J) Representative TIRF micrographs of a SUM159 cell expressing TKS5-mTagBFP2 as a marker for invadopodia and dTomato as a localization control for the dTomato-wGBD biosensor forming an invadopodia cluster after PDBu treatment (scale bar for both panels, 5 μm).

Cdc42 biosensor validation. (A) Representative micrographs of a SUM159 cell expressing the single-chain Cdc42 FRET biosensor and mCherry-cortactin as a marker for invadopodia after PDBu treatment (scale bar, 5 μm). The color calibration bar indicates the relative FRET ratio. White arrowheads indicate the invadopodia cluster; yellow arrowheads indicate the lamellipodia protrusion. (B) Quantification of FRET ratio and mCherry-cortactin intensity at individual invadopodia. Intensity and time have been rescaled between values of 0 and 1 for each invadopodia to allow for comparison. Lines are loess smoothing and 95% confidence from five invadopodia. (C) Representative micrographs highlighting Cdc42 FRET at invadopodia in a SUM159 cell as marked by mCherry-cortactin (scale bar, 5 μm). (D) Radial intensity analysis of FRET ratio and mCherry-cortactin intensity at invadopodia. Lines are expressed as mean ± SEM of 40 invadopodia. (E) Design of the Cdc42 activity biosensor used in this study, composed of the GTPase-binding domain of WASP (wGBD) tagged with dTomato. (F) Representative micrographs of SUM159 cells showing colocalization of the dTomato-wGBD sensor with different mTurquoise2-H2A-RhoGTPase-ΔCAAX constructs (RhoA-G14V, Rac1-G12V, or Cdc42-G12V; scale bar, 10 μm). (G) Quantification of the average intensity of dTomato-wGBD signal within the nucleus ratioed to the average intensity in the cytosol. Results are mean ± SD of at least 14 cells per condition from three independent experiments. Significance was tested with a two-tailed t test: ****, P < 0.0001. (H) Representative TIRF micrographs of lamellipodia protrusions from SUM159 cells expressing either dTomato as a control for cytoplasmic localization or dTomato-wGBD (scale bar, 5 μm). (I) Quantification of H in which a 10-pixel wide line was drawn perpendicular to the cell edge (yellow dashed line in H) and the intensity was measured for each frame. Distance 0 starts 10–15 μm within the cell and the protrusion can be visualized by a change in distance at intensity 0.25 over time. The increase in peak dTomato-wGBD intensity over time indicates an increase in Cdc42 activity localized to the leading edge. (J) Representative TIRF micrographs of a SUM159 cell expressing TKS5-mTagBFP2 as a marker for invadopodia and dTomato as a localization control for the dTomato-wGBD biosensor forming an invadopodia cluster after PDBu treatment (scale bar for both panels, 5 μm).

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