Cathepsin B surface expression and in vitro invasiveness. (A) Quantitative flow cytometry analysis of plasma membrane cathepsin B in PM1 and PM2 cell lines (empty histograms) and in MM1 and MM2 cell lines (full histograms) performed in cells found above (non invading) or below (invading) the Matrigel covered filter. Numbers represent the median fluorescence values ± SD among three independent measurements. Histograms from a representative experiment are shown. (B) Cytofluorimetric and fluorescence microscopy analyses of plasma membrane cathepsin B in MM4 cell line during selection of invading cells by repeated passages through the Matrigel-covered filters. (C) Cytofluorimetric analysis of plasma membrane cathepsin B in MM4 cells at the end of the selection after four passages through the Matrigel-covered filters revealed the presence of a sub-population with reduced size (R2 region, bottom central panel) and negative to cathepsin B (bottom left panel). In the bottom panels fluorescence microscopy micrographs showing cathepsin B-positive cells (corresponding to R1 region) and cathepsin B-negative microvesicles, probably corresponding to the sub-population (R2 region) revealed by cytofluorimetric analysis. Arrows indicate vesicles budding from (left picture) or near to (central picture) the cell. In the right panel a cluster of vesicles is shown.