Figure 5. 1,25-(OH)<sub>2</sub>D plus Dex dose response for increased mMCSF by RT-PCR. Semiquantitative RT-PCR for mMCSF and GAP was performed in controls and cultures exposed for 3 days to 1,25-(OH)<sub>2</sub>D (10 nm) and Dex (1, 10, and 100 nm). The phosphorimage top row shows RT-PCR for mMCSF, and the bottom row shows the RT-PCR for GAP. Lanes 1 and 2, Control cells; lanes 3–8, treated 10 nm 1,25-(OH)<sub>2</sub>D, with Dex added at 1 (lanes 3 and 4), 10 (lanes 5 and 6), and 100 nm (lanes 7 and 8). Each RT-PCR lane represents a separate collection of mRNA. Under the image, the quantitative phosphorimage data are shown below as mMCSF/GAP densitometry units averaged for two samples, with the control set at 100%. Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society
Figure 4. Dex regulates the mRNA for mMCSF isoform in ST2 cells Figure 4. Dex regulates the mRNA for mMCSF isoform in ST2 cells. A, Semiquantitative RT-PCR for mMCSF and GAP was performed in cultures exposed for 3 days to combinations of 1,25-(OH)<sub>2</sub>D and Dex as before (see Fig. 1). Experiments were compiled (n = 6) to show that Dex significantly increased the level of the mMCSF/GAP RT-PCR products. The addition of both Dex and 1,25-(OH)<sub>2</sub>D caused a further increase in the mMCSF/GAP product. Statistics: a≠ control, b ≠ control, and a, P < 0.05. B, The phosphorimage below the graph shows a representative experiment in which the labeled RT-PCR bands are visualized. The top row shows the RT-PCR product for mMCSF, and the bottom row shows the RT-PCR product for GAP. Lanes 1 and 2, Control; lanes 3 and 4, 1,25-(OH)<sub>2</sub>D; lanes 5 and 6, 100 nm Dex; lanes 7 and 8, 1,25-(OH)<sub>2</sub>D plus Dex. Each RT-PCR lane represents a separate collection of total mRNA. Under the image, the quantitative phosphorimage data are shown, represented as mMCSF/GAP densitometry units for each of the two samples, with the control set at 100%. Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society
Figure 3. Dex regulates MCSF expression in MOBC cells Figure 3. Dex regulates MCSF expression in MOBC cells. Dex (100 nm) was added for 3 days to cultures of MOBC cells. mMCSF was measured by bioassay (control, 5.9 ± 0.8 U/100,000 cells), and sMCSF was measured in the medium (control, 379 ± 47 U/100,000 cells by bioassay and 20 ± 5 U/100,000 by ELISA). Data were compiled from six experiments. Dex caused a significant increase (P < 0.05) in mMCSF alone (dark bars), but was not additive with 1,25-(OH)<sub>2</sub>D. Effects on sMCSF (light bars) did not reach significance by ANOVA. Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society
Figure 2. High levels of Dex suppress 1,25-(OH)<sub>2</sub>D-stimulated sMCSF in ST2 cells. ST2 cells were treated for 3 days with or without 1,25-(OH)<sub>2</sub>D (10 nm) with varying doses of Dex (Dex1, 1 nm; Dex10, 10 nm; Dex100, 100 nm; Dex alone, 100 nm). sMCSF in the medium was assayed by ELISA. sMCSF in control cultures was measured at 19.98 ± 4.6 U/100,000 cells. Data were compiled from at least six experiments; asterisks represent significant changes from control values (P < 0.05). Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society
Figure 1. Dex regulates MCSF expression in ST2 cells Figure 1. Dex regulates MCSF expression in ST2 cells. ST2 cells were treated as shown [1,25-(OH)<sub>2</sub>D, 10 nm; Dex, 100 nm] for 3 days. The cell layer was assayed for mMCSF by bioassay (dark bars), and sMCSF assayed by ELISA (light bars). mMCSF in control cells was 9 ± 2 U/100,00 cells compared with 511 ± 56 U/100,000 cells of bioassayable sMCSF. sMCSF measured by ELISA was standardized at 19.98 ± 4.6 U/100,000 cells. Data were compiled from at least six experiments; asterisks represent significant changes from control values (P < 0.05). Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society
Figure 6. Northern analysis for sMCSF in ST2 cells Figure 6. Northern analysis for sMCSF in ST2 cells. Total RNA was collected from ST2 cells treated for 3 days as specified, and Northern analysis was performed using [<sup>32</sup>P]CTP-labeled MCSF probe that hybridizes to the 4.3-kb mRNA encoding sMCSF. The top row shows phosphorimages of MCSF mRNA, and the bottom row shows the 18S of the ethidium bromide stain. The data are given below the figure as sMCSF/18S as a percentage of the control value (set at 100%). This Northern blot is representative of three similar experiments. Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society
Figure 7. Half-life of sMCSF in control and Dex-treated ST2 cells Figure 7. Half-life of sMCSF in control and Dex-treated ST2 cells. ST2 cells were treated for 3 days (with or without Dex). Actinomycin D (5 μg/ml) was added 10, 5, and 2.5 h before performing Northern analysis. The data were compiled from three experiments and represented as a decrease from 100% of mRNA in the untreated condition (no actinomycin) at the hour specified. Dex-treated cultures (without actinomycin) had 163% more mRNA for sMCSF than control cells in these three compiled repetitions. The sMCSF mRNA t<sub>1/2</sub> was 2.8 h in control cells, increasing to 7.5 h in cells treated with Dex. Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells* Endocrinology. 1998;139(3):1006-1012. doi:10.1210/endo.139.3.5778 Endocrinology | Copyright © 1998 by The Endocrine Society