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. 2021 Oct 27;118(44):e2114258118. doi: 10.1073/pnas.2114258118

Fig. 1.

Fig. 1.

Calcineurin stabilizes ER-α in MCF7 cells. (A) MCF7 cells were treated with the indicated concentrations of FK506 for 24 h, after which total cell lysates were prepared and subjected to immunoblot analysis with antibodies to ER-α and to β-actin (loading control). A representative immunoblot as well as the relative ER-α/β-actin band intensity ratio determined as the mean ± SEM from three independent experiments are shown. ***P < 0.001 and ****P < 0.0001 (Dunnett’s test). (B) MCF7 cells were treated with CN585 (0 or 15 μM) for 5 h, after which cell lysates were prepared and analyzed as in A. The quantitative data are means ± SEM from three independent experiments. *P < 0.05 (paired t test). (C) Lentivirus-infected MCF7 cells were cultured in the presence of Dox for 3 d to induce the expression of calcineurin A–α (shCaN A–α) or luciferase (shControl) shRNAs. Two different calcineurin A–α shRNAs (1, 2) were used. Cell lysates were then prepared and analyzed as in A, with the addition that the immunoblot analysis was also performed with antibodies to calcineurin A–α (CaN A–α). The quantitative data are means ± SEM from three independent experiments. **P < 0.01 and ***P < 0.001 (Dunnett’s test). (D) Cycloheximide (CHX) chase analysis of ER-α in MCF7 cells depleted of calcineurin A–α by exposure to Dox for 2 d, as in C. The Dox-treated cells were thus incubated with CHX (50 μg/mL) for the indicated times, after which cell lysates were subjected to immunoblot analysis with the indicated antibodies. The percentage of ER-α remaining after the various chase times was quantitated as mean ± SEM values from three independent experiments, and the half-life (t1/2) of ER-α was determined. *P < 0.05 and **P < 0.01 versus the corresponding value for calcineurin-depleted cells (unpaired t test). (E) MCF7 cells were treated with CN585 (30 μM) or MG132 (10 μM) for 6 h, after which cell lysates were analyzed as in A. Data are from a representative experiment. (F) Lentivirus-infected MCF7 cells were cultured in the presence of Dox for 3 d to induce the expression of control or calcineurin A–α shRNAs, after which total RNA was isolated from the cells and subjected to RT-qPCR analysis of ESR1 mRNA. Data are means ± SEM from three independent experiments. NS, not significant (unpaired t test).