Wnt10b stimulates osteoblast differentiation, we next investigated the purpose and appearance of Wnt6, Wnt10a and Wnt10b in the context of ST2 osteoblastogenesis. We examined the expression of the Wnt ligands during osteoblastogenesis in ST2 cells. Differentiation into osteoblasts was established by staining for matrix AP26113 mineralization with Alizarin red, and by elevated expression of alkaline phosphatase and osteocalcin, two osteoblast marker genes. Expression of Wnt6, Wnt10a and Wnt10b was noticeable during osteoblastogenesis, however, the degree of expression didn’t change during differentiation. These data declare that, in contrast to adipogenesis, transcripts for these Wnt ligands aren’t controlled all through ST2 osteoblastogenesis. Nonetheless, considering the fact that osteoblast differentiation is stimulated by Wnt10b, we next examined whether ectopic Wnt6 or Wnt10a also encourage osteoblastogenesis. We first analyzed whether ectopic Eumycetoma Wnts affect expression of genes associated with osteoblastogenesis before the induction of differentiation, to do this. As shown in Fig. 3A, ectopic Wnt10a or Wnt10b potently activated expression of alkaline phosphatase in ST2 cells. Ectopic Wnt6 also increased alkaline phosphatase expression, albeit to a much lesser extent than ectopic Wnt10a or Wnt10b. All of theWnt expressing cells also exhibited upregulation of Twist1, a factor thatmodulates osteoblastogenesis. Nevertheless, Wnt6, Wnt10a or Wnt10b did not notably influence expression of various other genes associatedwith osteoblast difference or activity. These cells were then induced to differentiate into osteoblasts and the degree of difference was determined by studies of matrix mineralization. This unveiled that Wnt10a or Wnt10b clearly influences osteoblastogenesis, with marked increases in Alizarin red staining and calcium content order Geneticin relative to EV cells. Wnt6 also triggered osteoblastogenesis, but, effects were weaker than those of Wnt10a or Wnt10b. These data show that Wnt6 and Wnt10a, like Wnt10b, can induce osteoblast differentiation. The above mentioned studies show that ectopic expression of Wnt6, Wnt10a or Wnt10b checks adipogenesis and influences osteoblastogenesis. But, whether endogenous expression of these Wnt ligands also modulates destiny of mesenchymal precursors remained to be identified. To analyze this possibility, ST2 cells were generated by us with shRNA mediated knockdown of Wnt6, Wnt10a or Wnt10b. All these Wnt ligands was significantly suppressed by appearance of the respective shRNAs. Wnt10b expression was also considerably reduced in the shWnt6 and shWnt10a cells, and Wnt6 expression was 80% lower in the shWnt10b cells, in keeping with mutual cross regulation of Wnt expression. Several technical difficulties were encountered by us in evaluating Wnt knockdown in these cell lines.