FIGURE 4.

Sirt1 potentiates TCF-mediated transcription by attenuating FoxO/β-catenin association. A, bone marrow-derived osteoprogenitor cells were transduced with a TCF-luc reporter construct and cultured in the presence of vehicle (veh) or SRT2104 for 24 h. RLU, relative luminescence units. B, GFP⁺ cell cultures derived from neonatal calvaria. C, top panel, osteoprogenitor cell culture lysates immunoprecipitated (IP) with an anti-β-catenin or -IgG antibody and probed with anti-FoxO1, anti-FoxO3, and anti-β-catenin antibodies. TL, total cell lysates; WB, Western blot. Bottom panel, relative amount of FoxO1 and FoxO3 in β-catenin immunoprecipitates. D, FoxO3 expression was induced in the OPF-iFoxO3 cell line by withdrawal of doxycycline, and cells were transfected with a TCF-luc reporter construct and with a Sirt1 expression plasmid, as indicated. Twenty-four hours later, vehicle or SRT2104 was added to the cultures for another 24 h. E, ST2 cells transfected with TCF-luc and with empty vector, FoxO1, or FoxO3 expression plasmids and co-transfected with Sirt1, as indicated. F, ST2 cells transfected with TCF-luc and with empty vector, wild-type (WT) FoxO1, acetylation mimic (KQ), or acetylation mutant (KR) and cultured in the presence of vehicle or 25 ng/ml Wnt3a for 24 h. G, acetylated FoxO1 (Ac-FoxO1) in bone marrow-derived osteoprogenitor cell cultures by Western blot. H, mRNA from vertebral bone homogenates of 8-week-old females. †, p < 0.05 versus respective vehicle; #, p < 0.05 versus vehicle in control group by two-way ANOVA. *, p < 0.05 by Student's t test. Bars represent mean and S.D. (error bars).