FIG. 6.

PTEN is a mediator of p53-induced suppression of Src phenotypes. (a to e) Effect of PTEN knockdown on the Src/Stat3/p53/caldesmon/podosome expression status in SMC-SrcY527F-wt p53 cells. (a) SMC-SrcY527F-wt p53 cells retrovirally transduced with anti-PTEN shRNAs. One clone each for shPTEN-1 and shPTEN-2 was analyzed by Western blotting. GAPDH was used as a loading control. (b to d) A stable SMC-SrcY527F-wt p53 cell line was transfected with one of the shPTEN expression constructs (coexpressing GFP) and was analyzed by immunofluorescence microscopy as indicated. TRITC-phalloidin was used for actin/podosome staining. Bars, 20 μm. (e) A stable SMC-SrcY527F-wt p53 cell line was transfected with either an empty vector (control) or one of two shPTEN expression constructs (shPTEN-1 and shPTEN-2) and was then assayed for podosome/rosette formation. Error bars represent standard deviations for three independent measurements. Asterisks indicate significant differences (P < 0.05) from the control. (f to i) Effect of PTEN overexpression on the Src/Stat3/p53/caldesmon/podosome expression status in SMC-SrcY527F cells. (f) SMC-SrcY527F cells were transiently transfected with increasing amounts of a wt PTEN expression construct (0, 4, and 8 μg DNA per 10-cm-diameter plate at about 80% confluence) using the Lipofectamine Plus protocol. Forty-eight hours posttransfection, samples were analyzed by Western blotting for proteins/phosphoproteins as shown. GAPDH protein levels indicate equal loading. (g to i) SMC-SrcY527F cells were cotransfected with wt PTEN and a GFP expression construct (pBabe-GFP) prior to analysis by fluorescence imaging. TRITC-phalloidin was used for actin/podosome staining. Bars, 20 μm. (j) PTEN mediates the suppressive effect of p53 on invasive phenotypes by inactivating Src/Stat3, which also serves to stabilize the podosome-antagonizing p53-caldesmon axis.