Figure 5.

Identification of the critical function of PTEN for survival under PNKP disruption. (A) Graphical representation of the forms of PTEN used to reconstitute PC3 cells, including the truncation mutation found in PC3 cells (R55fs*1), indicating the location of the other mutations in the phosphatase domain (C124S) and in the C2 structural domain involved in targeting proteins to cell membranes (K289E). The PDZ domain is a structural domain commonly found in signaling proteins, but was not mutated in these experiments (50). (B) Western blots of PC3 cells transfected with vector only (p.BABE.puro), cDNA for R55fs*1 or the different forms of full length PTEN. (C) Survival of PC3 cells transfected with expression vectors encoding various forms of PTEN and exposed to increasing concentrations of A12B4C3: p.BABE.puro (vector only); WT PTEN (full length) - wild-type PTEN; p.K289E – PTEN mutant with reduced nuclear shuttling; p.R55fs*1 – truncation mutant; p.C124S – phosphatase inactive PTEN mutant. Error bars represent ± SEM from at least three independent determinations, each experiment carried out in triplicate for a minimum of nine total assessed plates per point. (D) Survival response of PC3 cells transfected with a vector expressing wild-type RAD51 cDNA (WT RAD51) and exposed to increasing concentrations of A12B4C3. Error bars represent ± SEM from at least three independent determinations, each experiment was performed in triplicate for a minimum of nine total assessed plates. (E) Western blot of RAD51 expression in PC3 cells.