Figure 3

Effects of ascorbate and MAP kinase inhibitors on the Col X promoter and alkaline phosphatase activity in chick cephalic sternal chondrocytes. A: Activity of the b2/640 type X collagen promoter; 24 hrs after seeding cells were transfected with luciferase vectors, 5 hrs after transfection 4 μM U0126 or vehicle were added. Ascorbate was added to selected wells after a further hour. Values are mean ± S.D of the mean ratio of promoter to empty vector fluorescence units, for 3 experiments assayed in triplicate. B: Alkaline phosphatase activity; 24 hrs after seeding, medium was changed and 4 μM U0126 or vehicle was added. Ascorbate or ascorbate with BMP-2 was added to selected wells after a further hour. Cell extracts were prepared 72 hrs later. Data was obtained using 5 different isolates of chondrocytes assayed in triplicate. Values are mean ± SEM of 12–15 samples normalized to within experiment controls treated with BMP-2 but no inducers. *: p < 0.05 that group differs from non-supplemented group within inhibitor treatment. The increase in ALP caused by BMP-2 addition is shown, this increase is significantly smaller in UO126 treated cells, +:p < 0.05.