Fig. 2.

Shn3-deficient osteoblastic/mesenchymal cells are defective in driving osteoclastogenesis. (A) WT or Shn3^−/− cells were cocultured with WT BM osteoclast precursors in the presence of the indicated calciotropic agents. After 5 d, tissue culture supernatants were assayed for TRAP activity via colorimetric readout (A405). Error bars represent SD of absorbance from three independent wells. *P < 0.05. This experiment was repeated four independent times with similar results. (B) Representative photomicrographs of cocultures. (Magnification: 40×.) (C) RNA was harvested from cocultures, and expression of calcitonin receptor and cathepsin K was determined by quantitative real-time PCR assay. Levels of the indicated genes were normalized to actin and expressed relative to levels obtained with WT osteoblastic cells. Error bars represent SD of values obtained from PCR triplicate assays. This experiment was repeated three independent times with similar results.