Fig. 7. Effects of Bcl-2 and ZVAD on mitochondrial events associated with clusterin-induced apoptosis of HeLa cells by multiparametric analysis of HeLa/Bcl-2 cells by flow cytometry. Each cell is represented by a dot. The data are relative to GFP-SP-clu-, GFP--clu-, GFP-ß-clu- and GFP-transfected cells, overexpressing (b,e,h,k) or not (a,c,d,f,g,i,j,l) Bcl-2 and in the presence (c,f,i,l) or not (a,b,d,e,g,h,j,k) of ZVAD. For each sample, the mitochondrial membrane potential as measured by MitoTracker Red^TM fluorescence was reported in cytogram (MitoTracker Red versus size). Only GFP-positive cells with normal size were considered in multiparametric cytograms. Cells can be separated into two populations based on their MitoTracker Red fluorescence: one with high fluorescence (top) and the other with lower fluorescence (bottom). The proportion of cells in each population is specified in each panel of the cytograms (percentages correspond to only one experiment). The expression of GFP-clusterin constructs induced a loss of mitochondrial membrane potential, but cell size was not affected. (B) Proportion of variation of normally sized GFP-positive and MitoTracker-negative cells after Bcl-2 induction (black bars), ZVAD treatment (gray bars) or no Bcl-2 induction and no caspase inhibition (white bars). Values were calculated from three independent transfection experiments. Each bar represents the mean value±s.d. (n=3). ^*P<0.05 significantly different from control values; NS, not significantly different in Student's t-test.