Figure 2.

GH potentiates E2-stimulated T47D cell proliferation independently of IGF-I expression or IGF-IR activation. A, T47D cells were treated with 500 ng/mL GH for 24 hours. IGF-I mRNA expression was determined by QPCR. B, T47D cells were treated as in A, and conditioned media were collected. Levels of IGF-I protein were measured by ELISA. IGF-I (5 ng/mL) added to media served as a positive control. C, T47D cells were treated with hormones for 24 hours, after which total levels of IGF-IR protein were determined by Western blot. D, T47D cells were pretreated for 2 hours with 2 μM AEW541, an inhibitor of IGF-IR tyrosine kinase activity, followed by 30 minutes of treatment with GH+E2 or 50 ng/mL IGF-I. Phospho- and total IGF-IR levels were examined by Western blot to demonstrate the effectiveness of the inhibitor. E, T47D cells were treated with E2, E2+GH, or E2+IGF-I in the presence of 1 μM AEW541 or vehicle [dimethylsulfoxide (DMSO)] for 5 days. Proliferation was measured by the methylene blue assay. Similar results were found using the DNA assay (data not shown). Bars with different letters are significantly different (P < .05).