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. 2003 Jan 27;100(3):1034–1039. doi: 10.1073/pnas.0237312100

Figure 4.

Figure 4

Crosstalk between BMP-4 and estrogen signaling. (a) GH3 cells were treated with BMP-4, 17β-estradiol (E2), or their combination as indicated. After 72 h, cell proliferation was measured by WST-1 assay. As a control, cells were treated with TRH in combination with BMP-4, which, at the same saturating dose at which it did not interact with estrogen, produced a greater effect than did BMP-4 or TRH individually. *, P < 0.01 compared with basal values; ▴, P < 0.01 compared with 10 ng/ml BMP-4 or 1 nM 17β-estradiol individually; ■, P < 0.01compared with TRH or 200 ng/ml BMP-4 individually (ANOVA with Scheffé's test). (b) GH3 cells were treated with BMP-4, ICI 182780 (ICI), or their combination as indicated. After 72 h, cell proliferation was measured by WST-1 assay. The effect of TRH (used as a control) was not inhibited, indicating the specificity of the effect of ICI 182780. Similar results were obtained with tamoxifen. Bars represent the mean ± SE of quadruplicates from three independent experiments. *, P < 0.01compared with basal values; ▴, P < 0.01 compared with 200 ng/ml BMP-4 (ANOVA with Scheffé's test). (c) 17β-estradiol stimulation of cell proliferation was compared between GH3–vector and GH3–Smad4dn cells. Cells were treated with 17β-estradiol for 72 h, and cell proliferation was measured by WST-1 assay. PDGF stimulation was performed as a positive control of similar responsiveness between cell lines. 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 compared with the corresponding basal values (GH3–vector, 0.335 ± 0.011; GH3–Smad4dn, 0.395 ± 0.020); ▴, P < 0.01 between the two cell lines under the same treatment (ANOVA with Scheffé's test). (d) GH3 cells were treated with BMP-4, 17β-estradiol, ICI 182780, or their combination as indicated. After a 1-h treatment, cells were lysed, and the protein extracts were analyzed by Western blot for c-Myc as described in Methods. Equal loading was assessed by β-actin detection. One representative of three independent experiments with similar results is shown.