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. 2006 Mar 16;25(7):1456–1468. doi: 10.1038/sj.emboj.7601039

Figure 4.

Figure 4

Invadopodia in IMP-depleted cells. (A) Loss of actin-rich structures located at the cell–substratum interface. HeLa cells were transfected with Scr, IMP(1,3)A or IMP(1,3)B siRNAs, respectively, and stained with phalloidin to reveal F-actin-rich dot-like structures (arrows) resembling podosomes or invadopodia. (B) The percentile of cells containing podosomes or invadopodia after transfection with Scr, IMP(1,3)A, IMP(1,3)B or lamin A/C siRNAs was determined. Data are presented as the mean value±s.d. of three independent experiments. Asterisks depict statistically significant differences between the Scr siRNA-treated cells and the IMP(1,3)A and IMP(1,3)B siRNA-treated cells, respectively, by an unpaired, unequal t-test (***P<0.001). (C) HeLa cells were depleted for IMP and transfected with a mIMP1 expression plasmid—revealed by staining with anti-IMP1 (red)—which rescued podosome/invadopodia-like structures revealed by phalloidin staining (green). (D) Immunocytochemical analysis of tyrosine-phosphorylated proteins in the podosomes/invadopodia-like structures. HeLa cells were stained with Alexa Flour 488 conjugated phalloidin (green) and mouse anti-phosphotyrosine stained with Texas-Red conjugated anti-mouse antibody (red). The white box depicts the position of the blowups. Scale bar, 2 μm. (E) Immunocytochemical analysis of Arp2/3 complex protein p34-Arc and β1 integrin in the podosomes/invadopodia-like structures. HeLa cells were stained with phalloidin as above (green), in combination with rabbit anti-p34-Arc (red) and mouse anti-β1 integrin (blue) visualized with Texas-Red conjugated anti-rabbit antibody and Alexa Fluor 660 conjugated anti-mouse antibody, respectively (left panel). The white box depicts the position of the blowups (right panel). Scale bar, 2 μm. (F) Dynamics of podosome/invadopodia-like structures. HeLa cells were transfected with pEYFP-actin-expressing plasmid and actin turnover was examined by a FRAP analysis. The two upper pictures in the left panel show the area that was bleached before and after activation of the laser (white circle). The lower pictures show the recovery of EYFP-actin in the bleached area. The right panel shows the quantification of the recovery of EYFP-actin in the bleached area. The light intensity was measured in circular areas surrounding each of the four actin-rich structures present in the bleached area (blue circles). The green circle indicates the spot outside the bleached area that was used as reference. The results are stated as mean value±s.d. of the four blue circles in percentage of light emitted before bleaching. (G) ECM degradation assay. HeLa were seeded on an FITC-laminin-1-containing matrix, the cells were fixed and stained with Alexa Flour 660 conjugated phalloidin to depict the position of podosome/invadopodia-like structures. The dish was examined by confocal z-stacking, and the three lower pictures depict one of these slices, which show the colocalization between the matrix degradation spots and invadopodia structures. The upper right panel shows a vertical cross-section (lower right panel, blue line) of the cell and the underlying matrix with the projected invadopodia (arrows).