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Fig. S1. Invadopodium adhesion ring structures correlate strongly with ECM degradation. (A) Confocal images of SCC61 cells cultured on FITC-FN/1% gelatin and immunostained with antibodies recognizing the adhesion proteins paxillin (Pax), vinculin (Vinc), or active β1 integrin (12G10 mAb, “Active β1”) along with cortactin (Cort) or phalloidin to mark actin structures (Actin) or stably expressing GFP-α5 integrin (α5) and mCherry-Lifeact to mark actin structures. Arrows or zooms indicate adhesion protein rings surrounding invadopodia. CA1d cells cultured on FITC-FN were immunostained for phospho-Src (pY416) and cortactin (Cort). (B) Widefield epifluorescent images of SCC61 and CA1d cells plated overnight on “soft” or “hard” polyacrylamide (PA) gels or glass coverslips overlaid with fluorescent-labeled FITC-fibronectin (FN, green) and immunostained for actin (red) and cortactin (blue) to mark invadopodia. Rigidity values for soft and hard PA gels are reported as elastic moduli, calculated from rheometry measurements as previously described ( Parekh et al., 2011). (C) Percent of invadopodia associated with or without degradation, as indicated in SCC61 and CA1d cells. (D,E) Quantification of ECM degradation, invadopodia and focal adhesion (FA) characteristics on a per cell basis in SCC61 (D) and CA1d (E) cells. These data were used for the Spearman correlations shown in Fig. 1D. Box and whiskers indicate respectively the 25–75th and 5–95th percentiles with the dotted red line indicating the mean and the black line indicating the median. *P<0.05, **P<0.01, ***P<0.001. Scale bars = 5 µm.
Fig. S2. Effect of integrin-blocking antibodies on invadopodium activity and ring formation. SCC61 cells were treated with control antibody (20 µg/ml for A,B; 10 µg/ml for C,D) or inhibitory antibodies for β1 integrins (AIIB2, 10 µg/ml) or αvβ3 (LM609, 10 µg/ml). (A,C,D) Quantification of degradation area of FITC-FN/gelatin underneath SCC61 cells (B) Quantification of the number of paxillin-ringed cortactin containing invadopodia (Ringed), total cortactin invadopodia (Total), invadopodia localized with holes in the FITC-FN matrix (Active), and invadopodia localized with intact FITC-FN (Inactive) in SCC61 cells. *P<0.05; **P<0.01; ***P<0.001. n≥60 cells for each condition from 3 independent experiments.
Movie 1. SCC61 cells expressing Tom-Tractin (red, actin) and GFP-paxillin (green, adhesion) were imaged by confocal microscopy. Images were collected every 15 seconds with every other frame displayed at 10 frames per second for brevity. White boxes and “Actin” or “Pax” show timing of invadopodium formation and subsequent recruitment of GFP-paxillin to rings surrounding Tom-Tractin-positive invadopodia. Scale bar = 5 µm.
Movie 2. RGE control peptide-treated SCC61 cells imaged for Tom-Tractin (red, actin) and MT1-MMP-pHLuorin (green, MT1). Epifluorescent images were collected every 15 seconds with every fourth frame displayed at 10 frames per second for brevity. Deconvolved images are shown. White box outlines a cluster of invadopodia. Scale bar = 5 µm.
Movie 3. RGD integrin blocking peptide-treated SCC61 cells imaged for Tom-Tractin (red, actin) and MT1-MMP-pHLuorin (green, MT1). Epifluorescent images were collected every 15 seconds with every fourth frame displayed at 10 frames per second for brevity. Deconvolved images are shown. White boxes outline example invadopodia. Scale bar = 5 µm.
Movie 4. SCC61 cells expressing non-targeting control shRNA (NTC) and imaged for Tom-Tractin (red, actin) and MT1-MMP-pHLuorin (green, MT1). Epifluorescent images were collected every 15 seconds with every fourth frame displayed at 10 frames per second for brevity. Deconvolved images are shown. White boxes outline example invadopodia. Scale bar = 5 µm.
Movie 5. SCC61 cells expressing ILK1-targeting shRNA (ILK sh1) and imaged for Tom-Tractin (red, actin) and MT1-MMP-pHLuorin (green, MT1). Epifluorescent images were collected every 15 seconds with every fourth frame displayed at 10 frames per second for brevity. Deconvolved images are shown. White box outlines a cluster of invadopodia. Scale bar = 5 µm.
