FOXO overexpression in primary B cells causes cell-cycle arrest and increases apoptosis in a manner opposed by PI3K/Akt signaling. (A) Purified B cells from Balb/c mice were stimulated with 10 μg/mL LPS for 24 hours. The cells were then retrovirally transduced with either vector alone (pMIT), or wild-type or A3 mutant versions of FOXO1 (i,iii) or FOXO3a (ii,iv). (i-ii) Sixty-four hours after infection the cells were stained with anti-Thy1.1–biotin followed by Streptavidin-CyChrome to identify positively transduced cells and Annexin V–phycoerythrin (PE) to identify cells undergoing apoptosis. Bar graphs depict the percentage of apoptotic (Annexin V–positive) cells in the population expressing high levels of Thy1.1. (iii-iv) Thirty-six hours after infection, cells were stained with anti-Thy1.1–biotin and Streptavidin-FITC, then analyzed for DNA content by using propidium iodide. The percentage of live Thy1.1 cells in G1 (▪), S (▦) , and G2 (□) phases of the cell cycle was determined by using Flowjo. (v) A representative cell-cycle graph. Three to 4 independent experiments with similar results were obtained for both cell death and cell cycle. (B) Purified B cells from nontransgenic littermates (–Tg) and Bcl2 Tg (+Tg) mice were stimulated and retrovirally transduced as for panel A. Cell death (i-ii) and cell-cycle status (iii-iv) were monitored at 64 hours and 36 hours after infection, respectively, as for panel A.
