ERK is phosphorylated in HSCs of FoxO3a-deficient mice during hematopoietic recovery after 5-FU–induced myelosuppressive stress conditions. (A) (Left panel) Phospho-ERK immunostaining in BM mononuclear cells (unfractionated) or Lin⁻Sca-1⁺ cells 2 days after 5-FU injection in 8- to 12-week-old FoxO3a-deficient or wild-type mice. (Right panel) Data are mean frequency (%) plus or minus SD of phospho-ERK–positive cells in Lin⁻Sca-1⁺ cells (n = 5). *P < .01. Bar represents 100 μm. (B,C) FoxO3a-deficient cells are hyperresponsive to G-CSF (B) and IL-3 (C) and their phenotypes are restored by U0126 but not NAC treatment. Bone marrow mononuclear cells were isolated from wild-type and FoxO3a-deficient mice and cultured with 10 ng/mL G-CSF or 10 ng/mL IL-3 in methylcellulose medium for 7 days; then the number of colonies was scored. □ indicates wild-type cells (n = 5); and ■, FoxO3a-deficient cells (n = 5). Results are mean plus or minus SD of the number of colonies. *P < .01.