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. Author manuscript; available in PMC: 2009 Sep 10.
Published in final edited form as: Exp Cell Res. 2008 Jun 9;314(15):2884–2894. doi: 10.1016/j.yexcr.2008.06.003

Fig. 2. Limd1−/− calvarial osteoblasts display increased mineralization and support less osteoclastogenesis than Wt controls.

Fig. 2

(A) Primary Wt or Limd1−/− calvarial osteoblasts were cultured in normal or osteogenic media for 12 days then fixed and stained with Alizarin Red S to detect mineralization. The number of Alizarin positive nodules were counted and graphed. Data is a compilation of three different experiments, all performed in triplicate. For rescue experiments, Limd1−/− calvarial osteoblasts were infected with either myc-empty or myc-Limd1 retroviruses before exposure to osteogenic culture conditions. Aliquots of Limd1−/− cells infected with myc-empty or myc-Limd1 were lysed and equal amounts of protein run on SDS-PAGE then Western blotted for the presence of Limd1. (B) Immortalized Wt or Limd1−/− calvarial osteoblast were cultured in osteogenic conditions for 12 days then fixed, stained with Alizarin Red S, and the percentage of Alizarin Red S staining determined and graphed. (C) Equal numbers of primary Wt or Limd1−/− calvarial osteoblasts and wt BMDM were co-cultured in the presence of vitamin D for 10 days then fixed and TRAP stained to identify osteoclasts (white arrows). The number of TRAP positive osteoclasts was quantified and graphed. For rescue experiments, Limd1−/− calvarial osteoblasts were infected with either myc-empty or myc-Limd1 retroviruses before co-culturing. *Indicates a significant difference between Limd1−/− and Wt (*p<0.05, **p<0.01, ****p<0.001, student’s paired t-test).