Fig. 5. α-VE5 and γ-VE5- induced co-localization of Akt and PHLPP1 to the non-raft domains of the plasma membrane, essential role of PHLPP1 in VE5-induced Akt dephosphorylation, and in vivo tumor-suppressive activity of γ-VE5.

(A) Co-localization of Akt and PHLPP1 to non-raft membrane domains in LNCaP cells treated with α- and γ-VE5. Upper panels, Representative Western blot of PHLPP1, Akt phosphorylated at Ser⁴⁷³ (p-Ser⁴⁷³-Akt), Akt, PDK1 and flotillin-2 in cell membrane subfractions. Lower panels, A representative Western blot (left) and densitometric analysis (right) of the relative amounts of PHLPP1 and Akt phosphorylated at Ser⁴⁷³ in the non-raft fractions 10 - 12 from 3 independent experiments are shown (means ± S.D.). Signals from PHLPP1 and phosphorylated Akt were normalized to that of PDK1, the cellular distribution of which was unchanged by α- or γ-VE5 (Fig. 6C). Replicate data are shown in fig. S5A. *, P < 0.05 compared to respective DMSO control (Kruskal-Wallis). (B) Cholesterol content in individual membrane subfractions from cells described in (A). Means ± S.D. are shown (N = 3 independent experiments). *, P < 0.05 compared to the corresponding fraction in the DMSO control (one-way ANOVA). (C) Protective effect of siRNA-mediated PHLPP1 knockdown against the suppressive effects of γ-VE5 on cell viability (Left, The means and S.D. of 6 biological replicates from a representative experiment of a total of three independent experiments are shown), and on Akt phosphorylation at Ser⁴⁷³ (Right, A representative Western blot and densitometric analysis of relative phosphorylation of Akt at Ser⁴⁷³ from 3 independent experiments are shown) in LNCaP cells. Signal from phosphorylated Ser⁴⁷³ was first normalized to that of total Akt, and then to that of β-actin (means ± S.D.). Replicate data are shown in fig. S5B. *, P < 0.05 compared to respective DMSO control (Welch's ANOVA). (D) Effect of γ-VE5 on the growth of subcutaneo The cDNA fragments corresponding to the putative PH domain sequences of PHLPP1 us PC-3-luc xenograft tumors (mean starting tumor volume + SE, 75.3 ± 4.5 mm³). Left, Tumor burden as represented by relative bioluminescence (means ± S.D., n = 7 mice). Inset, representative tumors from vehicle- (Ctl) and γ-VE5-treated mice. P < 0.05 compared to control at 21 days (Student's t-test). Middle, Western blot analysis of the phosphorylation status of Akt at Ser⁴⁷³ (p-Ser⁴⁷³-Akt) and Thr³⁰⁸ (p-Thr³⁰⁸-Akt), MDM2, and IKKα/β in five representative tumors from each group. Right, Densitometric analysis of relative phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ in the representative tumors. Signals from phosphorylated Ser⁴⁷³ and Thr³⁰⁸ were first normalized to that of total Akt, and then to that of β-actin (means ± S.D., n = 5 tumors). *, P < 0.05 compared to control (Mann-Whitney U).