Expression of constitutively active Akt in 32D–EPO-R(d465) restores the ability of EPO to override γ-IR–induced growth arrest.

(A) Whole cell lysates of 32D–EPO-R(d465) clones stably expressing a Neo control plasmid, wild-type Akt, or myrAkt were Western blotted with anti-HA (αHA) antibodies. (B) Cells were left unstimulated (−) or were treated with IL-3 (1.4 ng/mL) or EPO (5 U/mL) for 8 minutes. Akt catalytic activity was assayed from cell lysates in an in vitro kinase assay (see “Materials and methods”). Alternatively, whole cell lysates were Western blotted with anti–phospho-Stat-5 (α P-STAT5) or anti–phospho-Erk (α P-Erk) antibodies. As a control for loading, lysates were probed with anti-Erk2 antiserum (α Erk). (C) Cells were cultured in medium containing either 1.4 ng/mL IL-3 or 5 U/mL EPO. Subsequently, cultures were exposed to 4 Gy γ-IR (+γ) or were left untreated (−γ). Twenty-four hours after irradiation, cells were stained with PI and analyzed by FACS. Before PI staining, cell viability was determined by trypan blue exclusion and was 80% or greater in all experiments. Percentage of S-phase cells is indicated for each culture. (D) Percentage of G2/M cells in γ-IR–treated cultures versus nonirradiated cultures shown in panel C was determined, and the fold increase in G2/M was calculated (G2+γ/G2−γ). Values presented represent the average of 3 independent experiments. Similar results were also obtained from polyclonal populations of each cell type. ■, IL-3; ▪, EPO.