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Fig. 9. Stimulation of ß-catenin signaling promotes osteoblast
differentiation. Whole calvarial (A, left panel) or CNC-derived (A, right
panel) osteoblast precursors, isolated from the Axin2+/+
and Axin2–/– littermates, were cultured in
differentiation media for up to 12 days. Lysates, isolated at different
differentiation days as indicated, were first analyzed by the alkaline
phosphatase liquid assay (shown in Fig.
4A,D). The same lysates were then used for immunoblotting (A).
Immunoblot analyses with the 
-ABC (activated) and
-ß-catenin (total) antibodies reveal that ß-catenin signaling
is significantly stimulated by inactivation of Axin2 during osteoblast
differentiation. The expression of FGFR1, which is increased upon
differentiation, is elevated in the Axin2–/– cells. The
level of actin was also analyzed as a control for protein content of the
lysates. (B,C) Quantitative real-time RT-PCR analyses were performed to
examine expression of two Wnt targets FGF4 and FGF18 in the CNC-derived
osteoblasts. The graphs represent the expression levels (in arbitrary units)
of FGF4 (B) and FGF18 (C) during the course of differentiation in vitro. (D)
The CNC-derived osteoblasts of Axin2+/+ and
Axin2–/– were cultured in differentiation
media for 7 days. The TOPFLASH reporter plasmid was then transfected by
lipofectamine with different combinations of the ß-catenin expression
(ß-cat) (Korinek et al.,
1997), ß-catenin RNA interference (ß-cat-shRNA)
(Cellogenetics) and pUC19 carrier plasmids, as indicated (0.5 µg for each
plasmid plus the carrier to a total of 1.5 µg DNA). Relative luciferase
activity (RLA) for each sample was determined after 48 hours. (E) Activation
of ß-catenin signaling by BIO stimulates osteoblast differentiation.
Primary osteoblast precursors isolated from nasal and frontal bones were
cultured in differentiation media with or without 2 µM BIO for up to 9
days. Liquid assays for alkaline phosphatase were performed at different time
points as indicated. The diagram is representative of three independent
experiments.
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