5a, that activation of endogenous p53 by doxorubicin increases PTEN expression and decreases the level of Stat3 pY705 in the two SMC and 3T3 cells, indicating that PTEN is a downstream effector of p53. Furthermore, Western blots showed that knockdown of PTEN by shRNA in smooth muscle cells coexpressing SrcY527F and wt p53 resulted in substantial increases inside the amounts of lively species of Src and Stat3, whereas the amounts of p53 and p53 inducible caldesmon and MDM2 have been de creased signi cantly during the very same cells. Photos of shPTEN transfected SMC SrcY527F wt p53 cells present that cells expressing shPTEN GFP expressed a greater degree of nu clear Stat3 plus a decrease degree of p53 than their nontransfected counterparts. Interestingly, PTEN knock down also led to abrogation within the suppression of your Src induced invasive phenotype by p53, as evidenced by the pres ence of significant numbers of podosomes rosettes in shPTEN expressing cells.
In contrast, we employed SMC SrcY527F cells to investigate whether the overexpression of wt PTEN alone may reverse the Src induced result on p53 and Stat3 expression and also the corresponding invasive phenotypes. Western blots show that overexpression of wt PTEN led to diminished amounts hop over to here of active Src and Stat3 and to elevated amounts of p53 and its in ducible gene solutions caldesmon and MDM2. This,nding is even more illustrated by,uorescence microscopy im ages, displaying that wt PTEN expressing cells possess a tremendously lowered nuclear Stat3 degree, an enhanced degree of p53, and consequently diminished podosome rosette counts. Statistical evaluation of those this content cells also shows that in excess of expression of wt PTEN impairs the means of SMC SrcY527F cells to kind podosomes. p53 stabilization has been shown for being a significant mech anism by means of which PTEN executes its tumor suppressive perform. The data presented in Fig. six indicate that PTEN mediated inactivation of proinva sive Src pY416 Stat3 pY705 also prospects to stabilization of the anti invasive p53 caldesmon axis.
These final results strongly impli cate PTEN because the mediator with the antagonistic result of p53 on Src Stat3 perform and Src Stat3 induced invasive phenotypes. The protein phosphatase activity of PTEN plays a dominant position in mediating the suppression of Src Stat3 perform and podosome formation. PTEN is actually a dual lipid

and protein phos phatase. Despite the fact that the lipid phosphatase action is well docu mented to play a major role in tumor suppression, recent information have implicated the protein phosphatase exercise of PTEN, as a result of a largely unknown substrate or pathway, while in the regulation of cell motility. To find out the contribution of your protein phosphatase activity of PTEN to the downregu lation of Src induced podosome formation, we generated two mutants, PTEN G129E and PTEN C124S, the former lacks lipid phosphatase activity but retains protein phosphatase ac tivity, when the latter is de cient in the two lipid and protein phosphatase routines.