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. Author manuscript; available in PMC: 2012 Oct 1.
Published in final edited form as: Bone. 2011 Jun 17;49(4):662–672. doi: 10.1016/j.bone.2011.06.010

Figure 5. Androgen-mediated opposite effects of ALP activity and mineralization during differentiation in cultures from AR3.6-tg fNCSC vs. pMSC.

Figure 5

Cultures were induced toward the osteoblast lineage with osteoinductive media and osteoblastogenesis assessed by ALP staining, alizarin red S (AR-S) and von Kossa staining. A. Characterization of osteoblastogenesis using ALP, von Kossa and AR-S staining. Both fNCSC and pMSC cells were grown with osteoblastogenic medium at confluence. At day 10, cultures were stained and mineralized nodule formation was assessed by von Kossa over ALP staining. For mineral accumulation analysis, both fNCSC and pMSC cells were grown with osteoblastogenic medium for 14 days, and mineralization was assessed by alizarin red-S (AR-S) staining. Similar analysis at lower power in 12-well plates is shown in right panel. B. AR-S quantitation after extraction. AR-S accumulation was expressed as fold vs. undifferentiated control. Data are expressed as mean ± SD (n = 3). Two-way ANOVA for the effects of genotype and embryonic lineage showed a significant interaction, so t-test was used to determine significance. **, p < 0.01 vs. WT; ##, p < 0.01 vs. fNCSC WT. Osteoblast markers ALP (C) and OC (D) gene expression. Levels were evaluated by qPCR using RNA isolated from mature mineralizing cells from both genotypes and both lineages grown in osteoblastogenic media for 10 days. Data are reported as mean ± SD (n = 3). *, p < 0.05; **, p < 0.01 compared to WT. UD = undifferentiated.