Figure 1. Caveolin-1 fails to accumulate at the trailing edge of migrating metastatic cells.

(A) Total protein extracts from DI-TNC1, MEF-3T3 and MDA-MB-231 cells were separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting with antibodies against caveolin-1 and β-actin. (B) Confluent monolayers of DI-TNC1, MEF-3T3 and MDA-MB-231 cells were wounded with a pipette tip and fixed at 0 (left panel) and 60 minutes (right panel) after monolayer injury. Cells were stained with a rabbit polyclonal anti-caveolin-1 antibody followed by an anti-rabbit IgG antibody (green) and nuclei were visualized with DAPI (blue). The first layer of cells facing the wounded area is distinguished from the rest by a white line. Scale bar, 20 µm. (C) Caveolin-1 distribution was evaluated by measuring the fluorescence intensity in four randomly chosen regions of equal dimensions at the front and the rear of the cell with the Image J software, as detailed in the Materials and Methods. Data represent the ratio of fluorescence intensity “rear/front” (mean ± SEM; n =  3). Statistically significant differences compared with time 0 minutes for each cell type are indicated (**, P<0.01; *, P<0.05). (D) B16-F10 cells transfected with either pLacIOP (mock) or pLacIOP-caveolin-1 (cav1) were treated or not with 1mM IPTG for 24 hours and total protein extracts were prepared, separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting. (E) Confluent monolayers of pLacIOP-caveolin-1 transfected B16-F10 cells in the absence or presence of 1mM IPTG were wounded with a pipet tip and fixed either at 0 or 60 minutes after wounding. Samples were stained with anti-caveolin-1 polyclonal antibody (green) and DAPI (blue). The edge of the wound is outlined with a white line. Scale bar, 20 µm. (F) Caveolin-1 localization was analyzed at different time points, as described in C. Data are the mean ± SEM from three independent experiments.