American Society of Hematology

Figure 3.

$Figure 3. IL-3 inhibits ALP and OC mRNA expression, early and late markers of OBL differentiation into osteocytes, but the inhibitory effects of IL-3 were not mediated by the Runx2 or β-catenin pathways. (A) RNA was isolated from primary murine stromal cells cultured for 10 days with increasing concentrations of IL-3. RT-PCR was performed with primers specific for murine ALP, OC, and Runx2. IL-6 was a negative control for ALP, OC, and Runx2 response. GAPDH was used as an internal control. (B) Primary human confluent pre-OB cells were treated with IL-3 (20 ng/mL) for 1 to 7 days. After the culture period, cell lysates were obtained and nuclear extracts were separated from the cytosolic fraction. Runx2 activity evaluation was determined by EMSA on nuclear extracts as described in “Materials and methods.” IL-3 did not change Runx2 activity in human cultures. Lysates were obtained after 24 hours or 7 days of IL-3 treatment in human stromal-cell cultures. (C) Whole cell lysates from human stromal-cell cultures treated with IL-3 for 24 hours or 7 days or from murine cultures for 24 hours or 6 days were prepared and subjected to Western blot analysis for β-catenin expression. β-actin served as an internal control.$

IL-3 inhibits ALP and OC mRNA expression, early and late markers of OBL differentiation into osteocytes, but the inhibitory effects of IL-3 were not mediated by the Runx2 or β-catenin pathways. (A) RNA was isolated from primary murine stromal cells cultured for 10 days with increasing concentrations of IL-3. RT-PCR was performed with primers specific for murine ALP, OC, and Runx2. IL-6 was a negative control for ALP, OC, and Runx2 response. GAPDH was used as an internal control. (B) Primary human confluent pre-OB cells were treated with IL-3 (20 ng/mL) for 1 to 7 days. After the culture period, cell lysates were obtained and nuclear extracts were separated from the cytosolic fraction. Runx2 activity evaluation was determined by EMSA on nuclear extracts as described in “Materials and methods.” IL-3 did not change Runx2 activity in human cultures. Lysates were obtained after 24 hours or 7 days of IL-3 treatment in human stromal-cell cultures. (C) Whole cell lysates from human stromal-cell cultures treated with IL-3 for 24 hours or 7 days or from murine cultures for 24 hours or 6 days were prepared and subjected to Western blot analysis for β-catenin expression. β-actin served as an internal control.

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