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. Author manuscript; available in PMC: 2010 Nov 6.
Published in final edited form as: Cell Stem Cell. 2009 Nov 6;5(5):527–539. doi: 10.1016/j.stem.2009.09.014

Figure 1. FoxO3 is expressed and active in NSC.

Figure 1

(A) FoxO3 is expressed in adult NSC in vivo. Immunohistochemistry on brain sections of 3 month-old mice with antibodies to FoxO3 (‘Ct’), Sox2, and NeuN in the dentate gyrus (DG) of the hippocampus and in the subventricular zone (SVZ) of. Sox2-positive/FoxO3-positive and NeuN-negative/FoxO3-positive nuclei are shown by white arrowheads. Higher magnification images are included. Scale bar: 100 μm.

(B) FoxO3 is expressed in purified NSC in culture. Immunocytochemistry on NSC isolated from 3 month-old mice with antibodies to FoxO3 (‘Ct’) and to Nestin. DAPI was used to stain nuclei. NSC were either grown as neurospheres (whole NS) (left panels) or freshly dissociated NSC (Diss. NSC) (right panels). Scale bar: 100 μm.

(C) FoxO3 expression in NSC at different ages. Western blots of protein lysates from secondary neurospheres in self-renewing conditions from FoxO3+/+ and FoxO3−/− littermates at 3 different ages, 1 day-old (1d), 3 month-old (3m), and 1 year-old (1y), probed with antibodies to FoxO3 (‘NFL’) and to β-actin. The data presented are representative of 3 independent experiments.

(D) FoxO3 phosphorylation is increased in differentiated progeny. Western blots of protein lysates of dissociated wild-type adult NSC in self-renewing conditions (day 0) or in differentiation conditions for increasing lengths of time (days 1-6) (Figure S3A). Western blots were probed with antibodies to FoxO3 (‘NFL’), to Phospho-T32 (FoxO3 T32-P) and to β-actin. Blots representative of 3 independent experiments.

(E) A FoxO-dependent reporter gene is responsive to FoxO3 in NSC. Luciferase assays in wild-type adult NSC using a promoter containing three FoxO binding sites driving the luciferase reporter gene (FHRE pGL3) or a control promoter (pGL3) and FoxO3 expression vectors (-: empty vector; WT: wild-type; CA: constitutively active; I: inactive). Values represent mean ± SEM from 3 independent experiments. One-way ANOVA, Bonferroni post-tests, *: p<0.05, **: p<0.01, ***: p<0.001.

(F) Endogenous FoxO3 is active in self-renewing NSC. Luciferase assays in FoxO3+/+ and FoxO3−/− adult NSC using FHRE pGL3 or control pGL3 as in E. Values were normalized to the first column. Values represent mean ± SEM from 3 independent experiments. One-way ANOVA, Bonferroni post-tests, **: p<0.01, ***: p<0.001.