FIGURE 4.

Statin-induced increased PTEN-pAkt binding correlates to PTEN ubiquitination. In A–F, cells were treated with insulin (1 μg/ml for 15 min) and thereafter with atorvastatin (1 μm for 1–5 min) as indicated. A shows cells subjected to PLA analysis. Purple dots indicate proximity between cellular bound antibodies for PTEN and pAkt Thr³⁰⁸. In several photographs, many dots are seen in or on the nuclear membrane. In B, columns summarize the total number of PTEN/pAkt Thr³⁰⁸ PLA signals in 50 cells 3 min after the addition of atorvastatin (using MATLAB), and in C, the distribution of pAkt/PTEN PLA signals between the nucleus and the cytoplasm in 50 cells is shown (each square represents a single cell). In D, lysates were immunoprecipitated (IP) with PTEN or α-calcineurin antibodies and analyzed for Akt or PTEN. Total lysates were analyzed for Cdk2. Error bars, S.D. from three independent experiments; *, significantly different from control, p ≤ 0.05. In E, cells were stained for ubiquitin. In F, a Western blot analysis employing PTEN antibody is shown. In G, cells were transfected with siRNA against NEDD4-1 (50 nm) for 72 h and thereafter treated with insulin (1 μg/ml for 15 min) and atorvastatin (1 μm for 5 min). Cells were stained for pAkt Thr³⁰⁸. The diagram shows the results expressed as the relative nuclear staining intensity (mean ± S.D. from 50 nuclei). *, significantly different from control; #, significantly different from insulin; **, significantly different from insulin and atorvastatin, p < 0.05. The Western blot in G shows a decreased level of NEDD4-1 in NEDD4-1 siRNA-transfected cells. In H, cells were pretreated with leptomycin B (10 ng/ml for 5 min) followed by insulin (1 μg/ml for 15 min) and thereafter with atorvastatin (1 μm for 5 min). Cells were stained for pAkt Tyr³⁰⁸. In I, cells were pretreated with MG132 (10 μmol/liter for 4 h) followed by insulin (1 μg/ml for 15 min) and thereafter with atorvastatin (1 μm for 5 min). Cells were stained for pAkt Tyr³⁰⁸.