Figure 3.

Human osteosarcoma cells are refractory to osteogenic BMP-induced ALP activity. (a) Endogenous expression levels of four osteogenic BMPs in human OS lines. Total RNA was isolated from the four OS lines and subjected to reverse transcription and quantitative real-time PCR analysis using primers specific for human BMP2, BMP6, BMP7, and BMP9 (listed in Supplementary Table 1). (b) Endogenous expression levels of early osteogenic genes Id1, Id2, and Id3 in human OS lines. Total RNA and quantitative real-time PCR analysis were carried out essentially similar to that described in (a), except using primers specific for human Id1, Id2, and Id3 (listed in Supplementary Table 1). All qPCR samples were normalized with the expression level of GAPDH. Each condition was done in triplicate. (c) Fold of changes in BMP2- and BMP9-induced ALP activity in the four OS lines. Subconfluent OS cells were infected with AdBMP2, AdBMP9, or AdGFP control. The infected cells were collected for ALP assay at the indicated time points. Each assay condition was done in duplicate. Fold of changes was calculated by dividing the ALP activity from BMP-treated samples with that of GFP-treated samples. The dotted lines indicate that the fold of change is 1.0 (ie, BMP-induced ALP activity is equal to that of GFP control-treated cells). See ‘Materials and Methods’.