Figure 2.

BMP-4 stimulates the proliferation of a lactotroph tumor cell line. (a) GH3 cells were treated with BMP-4, noggin (Nog), or their combination for 72 h. Cell proliferation was measured by WST-1 assay. Results represent the mean ± SE of quadruplicates from four independent experiments. *, P < 0.01 with respect to basal values; ▴, P < 0.01 with respect to 200 ng/ml BMP-4 stimulation (ANOVA with Scheffé's test). (b) Tumor explants were treated with BMP-4 or noggin (Nog) for 90 min, and c-Myc expression was analyzed by Western blot analysis as a parameter related to cell proliferation in different types of pituitary tumors (n = 9). PRL, human prolactinoma; GH, human GH-secreting pituitary tumor; NF, clinically inactive pituitary tumor. Equal loading was assessed by β-actin detection. (c) Cell proliferation under BMP-4 treatment was compared between GH3 cells stably transfected with an empty vector and GH3 cells with Smad4dn. Cells were treated for 72 h, and proliferation was measured by WST-1 assay. PDGF stimulation of GH3 cell proliferation was used as a positive control unrelated to BMP signaling. Bars represent the mean ± SE of the differences between the treated and the corresponding basal values of quadruplicates from three independent experiments. *, P < 0.01 with respect to the corresponding basal values (GH3–vector, 0.305 ± 0.015; GH3–Smad4dn, 0.365 ± 0.020); ▴, P < 0.01 comparing GH3–vector and GH3–Smad4dn cells under the same treatment (ANOVA with Scheffé's test). (Inset) Smad4dn (FLAG) expression was checked by Western blot analysis against a FLAG epitope contained in the GH3 cells with Smad4dn, as described in Methods.