FGD1 is enriched in collagenotytic invadopodia. (A) Upper panel: Cells transfected with TKS5^GFP (green) were seeded on type I collagen (gray) for 60 min and stained with FGD1 antibody (red) and DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. Lower panel: Correlation of FGD1 (x axis) and TKS5^GFP (y axis) pixel normalized fluorescence (Fluo) intensity along 16 invadopodial structures. inv, number of invadopodia analyzed; pix, total number of pixels analyzed for both markers. (B) Cells transfected with FGD1^GFP (green) were seeded on type I collagen (gray) and stained with TKS5 antibody (red), and nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. The lower panel shows the correlation of TKS5 and FGD1^GFP pixel fluorescence intensity as in A. (C) Cells seeded on type I collagen (gray) were stained for FGD1 (green) and cortactin (red) and with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. The lower panel shows the correlation of cortactin and FGD1 pixel fluorescence intensity as in A. (D) The gallery shows nonconsecutive frames from a representative video of a MDA-MB-231 cell expressing FGD1^GFP and TKS5^mCherry on a type I collagen fibrillary layer. The time-lapse sequence was started 45 min after plating the cells on type I collagen (see Video 1). Scale bar, 10 µm. (E) Immunoblotting analysis of MDA-MB-231, Hs578T and HT-1080 cell lysates with the indicated antibodies. Loading was controlled using actin antibody. (F–H) HT-1080 fibrosarcoma cells were plated on type I collagen (gray) and stained for the indicated invadopodia components. Scale bars, 10 µm; zoom-in of boxed regions, scale bars, 5 µm. The lower panels show the correlation of normalized fluorescence intensity for the indicated markers along the invadopodial structures as in A.