FIGURE 5.

Baicalin activates Wnt/β-catenin signaling pathway. A, total RNAs were extracted from cultured osteoblasts to perform PCR to determine the presence of the major components in the Wnt/β-catenin pathway, including the following: Wnt3a (435 bp), GSK-3β (356 bp), β-catenin (365 bp), and DKK-1 (148 bp) by using specific primers. PCR products were resolved on a 1% SYBR-stained agarose gel and visualized under UV light. The identities of PCR products were confirmed by DNA sequencing (data not shown). GAPDH (657 bp) served as an internal control. Representative images are shown, n = 3. B, baicalin induced the phosphorylation of GSK3β in a time-dependent manner. Baicalin (50 μm) or LiCl (10 mm) was applied onto cultured osteoblasts for different times as indicated. Total GSKβ and/or its phosphorylated form (P-GSK3β) (both at ∼46 kDa) was revealed by specific antibodies in a Western blot analysis (upper panel). The quantitation from the blots was shown by a densitometer (lower panel). Values are expressed as the fold of increase to basal reading (control culture treated with 0.02% DMSO). C, cDNA encoding GFP-tagged β-catenin was transiently transfected into cultured osteoblasts for 2 days. Baicalin (50 μm) or LiCl (10 mm) was applied onto transfected cultures for 4 h. The cytosolic and nuclear fractions were separated. The total cell lysate (upper panel) or nuclear fraction (lower panel) (both at 50 μl) was subjected to GFP fluorescent determination. D, treatment of baicalin in cultured osteoblasts was as in C. The nuclear fractions (at 0.5 and 2 h) were collected for Western blot analysis. The levels of β-catenin (∼95 kDa) and histone (a nuclear marker at ∼32 kDa) were revealed by specific antibodies. Values are expressed as the fold of increase to basal reading (control culture treated with 0.02% DMSO). In all cases, the values are in means ± S.E., n = 4, each with triplicate samples.