Fig. 1. Dominant-negative ezrin inhibits clonogenicity but modulates neither sensitivity to apoptosis nor HGF-mediated PKB/Akt stimulation. A, Immunoblot analysis for ezrin was performed using a polyclonal ezrin antibody. B,Immunoblot analysis for the VSVG-tagged NH2-terminal domain of ezrin (nter-ezrin, 43 kDa), and the VSVG-tagged mutant (F353-ezrin, 86 kDa) was performed using a VSVG antibody. C,Clonogenicity of control transfectants (open bars,left) or F353-ezrin (open bars,middle)- or nter-ezrin-expressing cells (black bars) was assessed by colony-forming assay (mean and SEM; n = 3; **p < 0.01;t test). D, Spontaneous DNA fragmentation of neo-, F353-ezrin-, or nter-ezrin-transfected LN-229 cells was assessed by DNA fluorometry (Wick et al., 1999a). As a positive control, nontransfected parental cells were treated with CD95L (50 U/ml) and CHX (10 μg/ml) for 16 hr (mean percentages and SEM;n = 3; *p < 0.05;t test). E, Neo (filled circles)-, F353-ezrin (filled squares)-, or nter-ezrin (open triangles)-transfected sublines of the LN-18, U87MG, LN-319, or LN-229 cell lines were treated with vincristine (VCR) for 72 hr (left panel), irradiated, and assessed for colony formation (middle panel), or treated with CD95L plus CHX (10 μg/ml) for 16 hr (right panel) (mean percentages; n = 3; SEM < 10%).F, PKB was immunoprecipitated from untreated (−) or HGF (10 ng/ml, 24 hr)-treated (+) LN-229 cells transfected with the neo control plasmid or nter-ezrin. The lysates were analyzed by SDS-PAGE and immunoblot analysis for PKB levels (top panel) and serine phosphorylation (bottom panel).