Fig. 4.

Macrophage-stimulating protein (MSP) may stimulate osteoblast differentiation via the RON receptor in the mesenchymal lineage C3H10T1/2 cells. (A) Effects of MSP on osteoblast-specific gene expression in C3H10T1/2 cells. Cells were cultured with MSP (100 ng/mL) for 3 or 9 days. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed with alkaline phosphatase (Alp), osteocalcin (Ocn), and osterix (Osx) primers. (B) Effects of MSP on ALP activity. C3H10T1/2 cells were cultured in an osteogenic medium in the absence or presence of MSP (+:50 and ++:100 ng/mL). After 10 days, the cells were subjected to ALP staining (upper panel) and ALP activity was quantified (lower panel). (C) Effects of MSP on calcium deposition. C3H10T1/2 cells were cultured as described above. After 3 weeks, the cell cultures were stained with alizarin red solution (upper panel). For quantitative analysis, the stained samples were treated with 10% cetylpyridinium chloride solution, and absorbance was measured by spectrophotometry (lower panel). (D) Effects of si-RON on MSP-induced gene expression. C3H10T1/2 cells were transfected with si-RON (50 nM) or control siRNA, and then MSP (100 ng/mL) was added. After 48 hr, cells were harvested, and qRT-PCR analysis was performed. *P<0.05 vs. control.