Figure 5

AA enhances LNShh cell-induced osteoblast differentiation. (A, B) MC3T3 cells were mixed with either LNCaP or LNShh cells then seeded onto 6-well plates. Mixed cultures were maintained for 14 days with or without exogenous AA and varying concentrations of DHP. Following, cultures were stained for ALP activity. In (B), cultures treated with AA in the presence or absence of 0.5 mM DHP were further examined under light microscopy to demonstrate staining for ALP activity in MC3T3 pre-osteoblasts. Magnification, 10x. Results are representative of 2 independent experiments. (C) LNShh cells were cultured with either MC3T3 cells that express the message for the human GLI1(−TAD) transgene (designated M-TAD cells) or parental MC3T3 cells as controls. Both MC3T3 and M-TAD cells express endogenous mouse Gli1 message; but, only the M-TAD cells express the dominant negative form of the transcription factor GLI1[6]. Mixed cultures were maintained for 7 days with or without AA and stained for ALP activity. Magnification, 10x. Results are representative of 2 independent experiments. (D) Mixed cultures were maintained for 7 days with or without exogenous AA. In this and subsequent figures, gene expression in MC3T3 cells in mixed cultures was determined by quantitative real-time RT-PCR analysis using mouse species-specific gene primers [6]. Expression of Akp2 in MC3T3 cells cultured with LNShh cells (filled bars) relative to those cultured with control LNCaP cells (open bars) were compared. Data from 2 independent experiments with similar results are presented.