Fig. 3.

Ligand-dependent and -independent activities of human GPR30 in T-REx HFF11 cells. (A) The treatment protocol of T-REx HFF11 cells for assaying GPR30 activity in (B), (C), and (E) is described. (B, C) T-REx HFF11 cells were treated without or with 1000 ng/ml TET for 12 or 24 hours. During the treatment without TET (−TET) or with TET (+TET), the cells were also treated without (no addition) or with vehicle or 0.1 μM PMA for 12 hours prior to assay for luminescence. The results are expressed as relative light units (RLU) and analyzed statistically using two-way ANOVA with Tukey’s multiple comparisons test (B), or as % of PMA values (C). (D) T-REx HFF11 cells were treated without (-ATP) and with 1 mM ATP (+ATP) for 12 hours prior to assay for luminescence. The results were expressed as RLU and analyzed statistically using an unpaired t test. (E) T-REx HFF11 cells grown in normal FBS (left graph) or in charcoal-treated FBS (right graph) for at least 2 days were treated without or with different concentrations of TET for 24 hours as indicated. During the treatment without or with TET, the cells were also treated with vehicle, 1 μM G-1 (G-1), 0.1 μM E2 (E2), or 0.1 μM PMA for 12 hours prior to assay for luminescence. The results are graphed as % of vehicle. The data are presented as means ± S.D. of at least three independent experiments. *P < 0.05; ***P < 0.001; ****P < 0.0001.