Fig. 5.

Calcineurin regulates ER-α activation. (A) Lentivirus-infected MCF7 cells were exposed to Dox in a medium containing charcoal-stripped serum for 3 d to induce the expression of control or calcineurin A–α (CaN A–α) shRNAs. The cells were then treated (or not) with 10 nM E2 for 4 h, after which total cell lysates were subjected to immunoblot analysis with antibodies to CaN A–αand to total or Ser¹¹⁸-phosphorylated (pS118) forms of ER-α. The relative ER-α/β-actin and ER-α–pS118/ER-α band intensity ratios were determined as mean ± SEM values from three or four independent experiments, respectively. *P < 0.05 and **P < 0.01 (paired t test). (B) MCF7 cells expressing the Dox-inducible luciferase (shControl) or shCaN A–α shRNAs were transiently transfected with an estrogen response element–luciferase reporter plasmid and then treated (or not) with E2 in a medium containing charcoal-stripped serum for 24 h, after which luciferase activity was measured in cell lysates. Data are means ± SEM from three independent experiments. *P < 0.05 (unpaired t test). (C) T47D cells that had been cultured in a medium containing charcoal-stripped serum for 2 d were incubated first with CN585 (30 μM) or dimethyl sulfoxide (DMSO) vehicle for 2 h and then in the additional absence or presence of E2 for 4 h. Total RNA was then isolated from the cells and subjected to the RT-qPCR analysis of expression of the indicated ER-α target genes. Data are means ± SEM from three independent experiments. ***P < 0.001 and ****P < 0.0001 (unpaired t test). (D) chromatin immunoprecipitation–qPCR (ChIP-qPCR) analysis of T47D cells treated as in C. After stimulation with E2 for 4 h, the cells were subjected to the ChIP-qPCR analysis of the binding of ER-α to promoter regions of its indicated target genes. Data are expressed as a percentage of the input chromatin and are means ± SEM of three independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired t test).