Table 1.
Primer sequences used for qRT-PCR.
Figure 1.
Mineralization and E11 expression by the MLO-A5 cell line.
(A) Alizarin red staining of MLO-A5 cultures over a 15-day time-course. (B) Quantification of Alizarin red staining by spectrophotometry. (C) E11 mRNA expression in the MLO-A5 cultures as determined by qRT-PCR. (D) Akp2 mRNA expression in MLO-A5 cultures. (E) E11 protein (∼38 kDa) expression by MLO-A5 cells. β-actin was used as a loading control. (F) Densitometric analysis of 3 independent E11 western blots. Results are mean±SEM (n = 3). * P<0.05, ** P<0.01, *** P<0.001 compared with previous time-point (B) or day 0 (C and F).
Figure 2.
Osteocytic differentiation of the MLO-A5 cell line.
The temporal expression patterns of the osteocyte-marker genes Dmp1 (A), Phex (B), Cd44 (C) and Sost (D) as assessed by qRT-PCR. Results are mean±SEM (n = 3). * P<0.05, ** P<0.01, *** P<0.001 compared to day 0.
Figure 3.
Osteocytic differentiation of primary calvarial osteoblasts in vitro.
The temporal expression patterns of the osteocyte marker genes, E11 (A), Dmp1 (B), Phex (C), Sost (D) and Cd44 (E) as assessed by qRT-PCR over 28 days of culture. (F) Mineralization of the primary osteoblast cultures as assessed by Alizarin red staining. Results are mean±SEM (n = 4). * P<0.05, ** P<0.01, *** P<0.001 compared to day 0.
Figure 4.
Inhibition of matrix mineralization in the MLO-A5 cell-line.
(A) Alizarin red staining of MLO-A5 cultures in which mineralization was inhibited by the addition of PPi or the omission of βGP and quantified by spectrometry (B). (C) Quantification of the collagenous matrix produced by the MLO-A5 cell after 9 days of culture as demonstrated by Sirius red staining (D) E11 mRNA expression in the mineralization-inhibited MLO-A5 cell cultures. (E) E11 protein expression in mineralization-inhibited and control MLO-A5 cultures as demonstrated by western blotting (F) Densitometric analysis of the protein expression at day 9 in 3 independent E11 western blots. Results are mean±SEM (n = 3). ** P<0.01, *** P<0.001 compared to day 0 (B) or AA & βGP (D and F) cultures.
Figure 5.
Effect of inorganic phosphate supplementation on MLO-A5 cultures.
Matrix mineralization (A) and E11 protein expression (B) after 9 days in control (AA and βGP) MLO-A5 cultures and cultures in which AA was omitted (βGP only). (C) Densitometric analysis of 3 independent E11 western blots. Results are mean±SEM (n = 3). ** P<0.01 compared to AA & βGP cultures.
Figure 6.
Effect of ECM mineralization inhibition on the expression of osteocyte and osteoblast marker genes in MLO-A5 cells.
Sost (A), Phex (B), Dmp1 (C), Cd44 (D), Ocn (E) and Col1a (F) mRNA expression in the mineralization-inhibited cultures as quantified by RT-PCR. Results are mean±SEM (n = 3). * P<0.05, ** P<0.01, *** P<0.001 compared to AA & βGP cultures.
Figure 7.
Effect of reversal of ECM mineralization inhibition on MLO-A5 cells.
(A) Alizarin red staining of MLO-A5 cultures in which mineralization was inhibited for 6 days before being promoted for a further 9 days. (B) Quantification of the results from (A) by spectrometry. Results are mean±SEM (n = 3). (C) E11 protein expression under the same conditions as (A) and (B). (D) Densitometric analysis of 3 independent E11 western blots. Results are mean±SEM (n = 3). *P<0.05, **P<0.01, ***P<0.001 compared to previous time-point.