TKS5 interaction with FGD1 is required for invadopodia formation. (A) Quantification of TKS5-positive invadopodia in siRNA-treated MDA-MB-231 cells. The y axis is the ratio of the TKS5 area to the total cell area normalized to the mean value of control siNT-treated cells (as percentage ± SEM). siNT, n = 127 cells; siFGD1, n = 146 cells, from three independent experiments. Mann-Whitney test. (B) Pericellular collagenolysis by MDA-MB-231 cells treated with the indicated siRNAs measured as mean intensity of Col1-^3/4C signal per cell. Values for control cells were set to 100%. siNT, n = 34 cells; siFGD1, n = 30 cells; siTKS5, n = 28 cells, from three independent experiments. Kruskal-Wallis test. (C) Position of W-to-A substitutions in TKS5 SH3^#4 (W861A) and SH3^#5 (W1092A) domains. (D) MDA-MB-231 cells were knocked down for endogenous TKS5 upon siRNA treatment, and transfected with the indicated TKS5^GFP constructs. Cells were plated on type I collagen (gray) and stained for GFP tag (green) and FGD1 (red) and nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, scale bar, 5 µm. (E) TKS5^GFP/FGD1 colocalization map (magenta) in the different cell populations as in D. The underlying collagen network is shown in blue. Scale bar, 10 µm. (F) TKS5^GFP/FGD1 Manders correlation coefficient in the different cell populations as in D. Tukey’s multiple comparisons test. (G) Quantification of FGD1-positive invadopodia in cells silenced for endogenous TKS5 as in D and transfected with the indicated TKS5^GFP constructs as in D. The y axis is the ratio of the FGD1 area to the total cell area normalized to the mean value of control siTKS5/TKS5^GFP-treated cells (as percentage ± SEM). TKS5^GFP, n = 47 cells; W861A, n = 50 cells; W1092A, n = 53 cells; W861A+W1092A, n = 43 cells from three independent experiments. Tukey’s multiple comparisons test. (H) MDA-MB-231 cells were knocked down for endogenous FGD1 upon siRNA treatment, and transfected with the indicated FGD1^GFP constructs. Cells were plated on type I collagen (gray) and stained for Tks5 (red) and nuclei were stained with DAPI (blue). Scale bar, 10 µm. (I) Quantification of TKS5-positive invadopodia in the different cell populations. The y axis is the ratio of the TKS5 area to the total cell area normalized to the mean value of control siNT-treated cells (as percentage ± SEM). siNT, n = 75 cells; siFGD1, n = 74 cells; siFGD1/FGD1^GFP, n = 98 cells and siFGD1/FGD1^GFPΔPRD, n = 67 cells from three independent experiments. Tukey’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001; ns, not significant.