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. 2009 Aug 24;186(4):571–587. doi: 10.1083/jcb.200812176

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© 2009 Oser et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Invadopodium precursors form in response to EGF and mature to degrade matrix. (A and B) MTLn3 cells expressing GFP-actin were cultured on Alexa 568-FN/gelatin thick matrix and analyzed by time-lapse microscopy. (A) Formation of the precursor before degradation is shown (arrowhead). Bars, 10 μm. (B) Quantification of GFP-actin fluorescence intensity at all stages of invadopodia (blue) vs. that of underlying matrix (red). n = 3 invadopodia; three independent experiments. (C and D) Live cell time-lapse experiments using MTLn3 cells expressing TagRFP-cortactin plated on thin 488-gelatin matrix. (C) Representative montage showing the formation of an invadopodium precursor (top) precedes gelatin matrix degradation (bottom). 3 min/frame. Bar, 1 μm. (D) Quantification of the change in cortactin fluorescence intensity at invadopodia vs. that of the underlying matrix in response to EGF. n = 25 invadopodium precursors, two independent experiments. For cortactin, P < 0.05 for all times compared with 0 min. For 488-gelatin, P < 0.05 for all times compared with 0 min, except 1 min where P > 0.05. (E) Representative images of cortactin and Tks5 antibody staining of MTLn3 cells stimulated with EGF. Arrowheads indicate invadopodium precursors. Bar,10 μm. (F) Quantification of the number of invadopodium precursors per cell that contain Tks5 and cortactin after EGF stimulation. n = 40 (0 min), 37 (1 min), and 17 (3 min) cells; three independent experiments. P values are compared with 0 min. (G) Image showing that Tks5/cortactin punctate structures colocalize with matrix degradation (arrowheads). (H) Representative montage (background subtracted) showing that TagRFP-cortactin (top) and GFP-MT1-MMP (bottom) colocalize at invadopodium precursors formed in response to EGF. 20 seconds/frame. Bar, 1 μm. Quantification of (I) fluorescence intensity and (J) rate of fluorescence intensity increase of TagRFP-cortactin or GFP-MT1-MMP at invadopodium precursors formed in response to EGF. n = 33 invadopodium precursors; three independent experiments. For I, P < 0.05 for all times compared with 0 min.