Inhibition of PI-3K–dependent signaling pathways prevents IL-3 and EPO from overriding γ-IR–induced growth arrest.

(A) 32D–EPO-R(wt) cells were cultured in medium that contained either 1.4 ng/mL IL-3 or 5 U/mL EPO. Cultures were then supplemented with either DMSO (0.2%) or LY294002 (10 μM). 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, viability of the cells was determined by trypan blue exclusion and was 94% or greater in all experiments. Percentage of S-phase cells is indicated for each culture. (B) Percentage of G2/M cells in γ-IR–treated cultures versus nonirradiated cultures shown in panel A was determined, and the fold increase in G2/M was calculated (G2+γ/G2−γ). Values presented represent the average of 3 independent experiments. ■, DMSO; ▨, LY294002. (C) 32D–EPO-R(wt) cells treated with either DMSO (0.2%) or LY294002 (10 μM) were left unstimulated (−) or were stimulated with 1.4 ng/mL IL-3 or 5 U/mL EPO 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-Erk (α P-Erk) or anti–phospho-Stat-5A/B (α P-STAT5) antibodies. As a control for loading, lysates were probed with anti-Erk2 (α Erk) antiserum.