Figure 3. Gene expression analysis of short-time receptor stimulus and short-time Smad-phosphorylation.

(A) 30 h experiment using the stable reporter cell line. The cells were stimulated with 0.1 nM (green), 1 nM (red) 10 nM (blue) BMP2 or non-stimulated (black) and after 15 minutes the stimulation medium was removed and fresh starvation medium were given to the cells. Then every hour 50 µl medium was withdrawn and the Luciferase activity was measured. The relative fold change to the unstimulated control is depicted. (B) Quantitative real-time PCR on id1 and smad6. The cells were stimulated with 0.1 nM (green), 1 nM (red) BMP2 or non-stimulated (black) for 15 minutes and then cultivated in starvation medium until cell lysis. (C) 30 h experiment using the stable reporter cell line. The cells were stimulated with the indicated ligand concentrations and after 15 minutes Dorsomorphin was added to the cells. 50 µl medium were withdrawn every hour and the Luciferase activity was measured. The relative fold change to the unstimulated control was calculated and assigned. (D) qRT-PCR analysis of id1 and smad6. The cells were stimulated with the indicated concentrations for 15 minutes and Dorsomorphin was given to the cells. Every hour one sample was lysed and frozen at −80°C until the further processing.