Figure 4

Immunohistochemical staining and quantitation of anoikis in PC-3M cells. A. uPAR over-expression and apoptosis in PC-3M suspension culture. Thin arrow indicates a fluorescence overlay of a live cell (blue) which over-expresses uPAR (green). The cell surface uPAR expression is characterized by a stippled staining pattern in the central region of the cell and forms a peripheral hallow. This uPAR+ cell is easily distinguishable from a live cell with wild type-uPAR expression (wide arrow). Apoptotic cells are shown with nuclear up take of orange-red staining from ethidium bromide. The nuclei demonstrate various degrees of condensation while the cell body show no up take of Calcein blue. B. The percentage of apoptosis in cells with wild-type (□) and uPAR+ (▲) are plotted over a 3 day period of suspension. Apoptosis in individual cells was defined as cells which allowed ethidium bromide to permeate and stain the nucleus (indicative of a breached plasma membrane) and were unable to accumulate Calcein blue methoxyester (fluorescent marker for live cells). uPAR over-expressing cells were identified by immunofluorescent staining using Alexa Fluor 488. Data represent mean ± S.D. of four replicate samples.