Figure 1.

Lmx1b overexpression inhibits osteoblast differentiation and function. Primary osteoblast precursor cells were cultured in OGM (osteogenic medium) for six days. (a) Total RNA was isolated from cell lysates at the indicated times, and the mRNA expression levels of Lmx1b, Runx2, alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), and osteocalcin (Bglap) were determined using quantitative real-time PCR. Data represent the means ± SD of triplicate samples. ** p < 0.001, # p < 0.005 vs. day 0. Values shown are normalized to Gapdh levels. Data represent means ± SD of triplicate samples. ** p < 0.001, # p < 0.005 vs. 0 day. Results are representative of experiments that were independently repeated at least three times. (b–e) Primary osteoblast precursor cells transduced with pMX-IRES-EGFP (Control) or Lmx1b retrovirus were cultured in OGM. (b) Cells cultured for four days were analyzed for alkaline phosphatase (ALP) activity. Data represent the means ± SD of triplicate samples. * p < 0.05 vs. control. (c) Cells cultured for six days were fixed and stained with alizarin red. Alizarin red staining activity quantified by densitometry at 570 nm. Data represent the means ± SD of triplicate samples. * p < 0.05 vs. Control. (d) Total RNA was isolated, and the mRNA expression levels of Lmx1b, Runx2, Alpl, Ibsp, and Bglap were determined using quantitative real-time PCR. Data represent the means ± SD of triplicate samples. * p < 0.05, # p < 0.005 vs. control. Results are representative of experiments that were independently repeated at least three times. (e) Cell lysates obtained at each time point were analyzed using western blotting with specific antibodies, as indicated.