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. 2016 Feb 1;27(3):451–465. doi: 10.1091/mbc.E15-09-0615

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© 2016 Bleaken et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — CD44/vimentin-rich mesenchymal cells that express markers of invadosomes are inherently invasive in Matrigel-Transwell chemoinvasion assays. (A–C) At 2 d in culture, cells that have invaded to the bottom of Matrigel-coated Transwell filters were coimmunostained for vimentin and CD44. Identification of these invaded cells as mesenchymal was determined by their positive labeling for both CD44 (A, C; red) and vimentin (B, C; green). Bar, 20 μM. (D–L) Cells that invaded in the Matrigel-Transwell chemoinvasion assays were labeled for characteristic invadosome molecules on day 2. Invaded cells were coimmunostained for the following combinations: cortactin (D, F; red) with Tks5 (E, F; green); MMP-2 (G, I; red) with MMP-9 (H, I; green); or cortactin (J, L; red) with MMP-9 (K, L; green). Invaded cells were rich in all invadopodia molecules examined, and regions of colabeling for cortactin and Tks5 (D–F, arrows), as well as cortactin and MMP-9 (J–L, arrows) were evident where these molecules may function in invadosomes for invasive function. Bar = 10 μm (D–I), 5 μm (J–L). (M, N) A Matrigel-Transwell chemoinvasion assay at 24 h in culture labeled for vimentin (green) and F-actin (blue) and also imaged by differential interference contrast (DIC). (M) A single vimentin-rich mesenchymal cell that had invaded through Matrigel to the bottom of the filter through a pore in the filter (seen as a hole in the DIC image; white arrow) is imaged en face, represented by the red arrow. Bar, 50 μm. (N) The same cell (red arrow) as shown in a 3D reconstruction side view of confocal Z-stacks collected from the explant basement membrane capsule of the inverted explant on top of the filter through to the cells that had invaded to the bottom of the filter. Here the transit of this cell through the filter pore (white arrow) is evident. Bar, 30 μM. Here A–C and M–N are Matrigel-Transwell chemoinvasion assays in which the explant on top of the filter was not removed before immunostaining. In D–L, the explant on top of the filter was removed before labeling.