Fig. 1. Tocopherols induce apoptosis and the Ser⁴⁷³-specific dephosphorylation and membrane localization of Akt.

(A) Chemical structures of α- and γ-tocopherol (upper panel) and their effects on the viability of LNCaP (LN) and PC-3 cells (lower panel). Points, means; bars, ± S.D. (n = 6 replicates; data from a representative experiment). α, α-tocopherol; γ, γ-tocopherol. (B) Flow cytometric analysis of apoptosis in LNCaP cells measured by annexin V (FL1-H) and PI (FL2-H) staining after exposure to α-tocopherol (α-Toco) and γ-tocopherol (γ-Toco). Representative dot plots and the percentages of cells that were apoptotic from 3 independent experiments are shown (means ± S.D.). *, P < 0.05 compared with the DMSO control (one-way ANOVA). (C) Western blot analysis of the effects of α- and γ-tocopherol on the phosphorylation of Ser⁴⁷³- and Thr³⁰⁸-Akt (p-Ser⁴⁷³-Akt and p-Thr³⁰⁸-Akt) in LNCaP cells. A representative Western blot and relative phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ from 4 independent experiments are shown. Densitometric signals from phosphorylated Akt were first normalized to that of total Akt, and then to that of β-actin (means ± S.D.). Replicate data are shown in fig. S1B. *, P < 0.05 compared with the DMSO control (Kruskal-Wallis). (D) Plasma membrane localization of Akt in LNCaP cells exposed to α- and γ-tocopherol. Left, Immunofluorescence staining of Akt (red) and DAPI (4′,6-diamidino-2-phenylindole)-stained nuclei (blue). Scale bars, 40 μm. Arrows indicate cells selected for analysis of fluorescent intensity through a cross-sectional plane (center, yellow line). Right, Three-dimensional surface plots of whole cell fluorescence intensities. x, y, and z axes, μm. Two-dimensional histograms show cross-sectional fluorescence intensities. Replicate data are shown in fig. S1D. (E) Percentages of cells that exhibited membrane of Akt in the experiments described in (D) are shown. *, P < 0.05 compared with DMSO control (one-way ANOVA; 73 - 142 cells were analyzed for each treatment condition in 3 independent experiments).