Figure 6. Delayed early stage osteoblast differentiation in Morc3^{mut +/−} mice.

Osteoblasts from the long bones of 12 week old WT and Morc3^{mut +/−} mice were cultured in osteogenic media with dexamethasone (10 nM), β-glycerophosphate (10 mM) and ascorbate (50 μg/ml). (A) Representative images of alizarin red S stained mineralised nodules. (B) Quantitative analysis of mineralised nodule area expressed as the relative % of wild type control (n = 6). (C) Alkaline phosphatase activity (units per mg of protein; U/mg) was measured in whole cell lysates from osteoblast cultures at day 0, 7 and 21. (n = 3) (D–I) Real Time-PCR analysis of gene expression in Morc3^{mut +/−} long bone osteoblast cultures expressed as fold change relative to wild type at day 0 control; (D) Morc3, (E) Rankl/Opg (expression ratio), (F) Osteocalcin (Bglap1), (G) Alkaline phosphatase (Alpl), (H) Stat1, and (I) Ifnb1. Hprt1 was used as integral housekeeping control. (J) Representative western blot image of Morc3, STAT1, Rankl, OPG and β–catenin protein levels during WT and Morc3^{mut +/−} osteoblast differentiation. (K,L) Quantitative analysis of protein levels by densitometry, bands were normalized to β-actin loading control and compared to WT day 0 control (n = 3); (K) Rankl/OPG (expressed as a ratio) and (L) β-catenin. Data are presented as fold change ± SEM. NS = non-significant; *p < 0.05; **p < 0.01.