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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: J Cell Physiol. 2020 Jan 27;235(10):6673–6683. doi: 10.1002/jcp.29563

Figure 3. Fzd4 is required for normal osteoblast differentiation and Wnt signaling in vitro.

Figure 3.

(A) qPCR analysis of Fzd4 mRNA levels in control and ΔFzd4 osteoblasts. (B) Assessment of osteoblast proliferation by flow cytometric analysis of BrdU incorporation. (C) Alkaline phosphatase and Alizarin red S (ARS) staining of cultures of control and ΔFzd4 osteoblasts cultured for 14 days under osteogenic conditions. (D) Quantification of calcium deposition via the extraction of ARS stain. (E and F) qPCR analysis of osteogenic markers (E) and regulators of osteoclastogenesis (F). (G) qPCR analysis of inhibitors of matrix mineralization. (H) Quantification of pyrophosphate (PPi) in cultures of control and ΔFzd4 osteoblasts. (I) qPCR analysis of Wnt ligand mRNA levels in control and ΔFzd4 osteoblasts (n.d., not detected). (J) Fold increase in Axin2 mRNA levels after Wnt3a (100ng/ml) treatment of differentiating control and ΔFzd4 osteoblasts. (K) Western blot analysis and quantification of β-catenin protein levels in control and ΔFzd4 osteoblasts left untreated (NT) or treated with Wnt3a. (L) Representative microCT images of the distal femur of 12 week old control and Ndp−/y mice and quantification of trabecular bone volume (n=9–11mice/genotype). All results are expressed as mean ± SEM. *, p<0.05.