Tamoxifen

If not otherwise stated all compounds were obtained from Sigma-Aldrich. For in vitro studies 5 μM cyclopamine (Toronto Research Chemicals Inc.) dissolved in ethanol (EtOH), 10 μM tamoxifen in DMSO and 1 μg/ml recombinant murine Shh-N (rShh-N; R&D systems) in HD-buffer were used. tamoxifen solution for in vivo use was prepared as described (see below)23 (link).

Primary calvarial osteoblasts from CAGGCre-ER-Rspo3^flox/flox and Rspo3^flox/flox control mice were isolated as above and cultured in complete α-MEM for 2–3 days prior to re-seeding in multiwell plates and culture in complete osteogenic media. Cells seeded in 48-well plates were cultured in osteogenic media for 24 h, followed by addition of tamoxifen (Sigma, ReadyMade solution, SML1666) for additional 24 h, before complete media change and continued culture in complete osteogenic media without tamoxifen. 50 or 100 nM tamoxifen was used and resulted in similar results. Osteoblastic differentiation was followed and analyzed as described above with the time of tamoxifen removal designated day 0. PTH (10 nM, Bachem, H-1660) and PGE2 (1 µM, R&D Systems, 2296) was added the day after tamoxifen removal (day 1) and continued until day 6, when cells were harvested for gene expression analysis as described above.