GPR30 mediates the effects of 17β-estradiol on hDF shape.

<p><b>a.</b> Efficiency of shRNA vectors. hDF were transfected with shGPR30 (#1 and #2) or control (shC). Expression of the indicated proteins was determined 48 h after transfection. <b>b.</b> ERK phosphorylation under the indicated conditions. Ratios of phosphorylated ERK (p-ERK) to total ERK (t-ERK) are indicated. Lower panel: quantification of the western blots performed on the four donors. Data are presented as mean +/- s.e.m. relative to vehicle-treated control. <b>c.</b> Cells shape monitored under the indicated conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120672#pone.0120672.g001" target="_blank">Fig. 1b</a>. n = 400 cells. ns: not significant; ***<i>p</i><0.001.</p

17β-estradiol treatment correlates with focal adhesion strengthening.

<p><b>a.</b> Confocal pictures of donor 1 cultured in the presence of FCS, or DS supplemented or not with 10^-7 M E2. Vinculin (green), actin (red) and nuclei (blue) were stained. <b>b.</b> Quantification of focal adhesions number, related or not to cell area or cell perimeter. <b>c.</b> Quantification of vinculin staining intensity and adhesive area by cell. <b>d.</b> Diagram of jitter plot (left) and mean (right) describing the distance of vinculin staining to the periphery of the membrane. Diagrams (except jitter plot) represent the mean±SEM of n>50 cells per condition. ***: p<0.001, ns: not significant.</p

17β-estradiol remodels cell shape in dermal fibroblasts.

<p><b>a</b>. hDF were cultured in the presence of untreated (FCS) or desteroided serum (DS) or in DS-containing medium supplemented with vehicle (DS) or 10–7M 17β-estradiol (DS+E2). Bright field microphotographs with cells from donor 1 are shown. <b>b-c</b>. Cells were cultured as indicated, analyzing kinetics of E2 exposure (<b>b</b>) or dose-response (<b>c</b>). Quantifications were performed indicating the ratio between the longest to shortest axis. Diagram represents mean±SEM of 400 cells quantified on 4 independent donors. 100 cells per condition were counted for each one donor. *p<0.05; ***p<0.001. Scale bars = 100 μm.</p

17β-estradiol effect on cytoskeleton re-organization involves non-genomic mechanisms.

<p><b>a.</b> Cells were cultured in the presence of FCS, or DS supplemented or not with 10^-7 M E2 and cycloheximide (CHX; 20 μg/ml). Actin and nuclei were stained. Scale bar = 100 μm. Lower panel: expression of the ERRα protein upon treatment with CHX for the indicated time was analysed by western blot with actin used as a loading control. <b>b.</b> Cells were cultured in the presence of DS for 2 days, then treated with 10^-8 M E2 or its membrane-impermeable conjugate (E2-BSA). Cell shape was monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120672#pone.0120672.g001" target="_blank">Fig. 1b</a>. n = 400 cells per condition. Values are mean +/- s.e.m; ns = not significant, ***<i>p</i><0.001.</p

Pharmacological modulation of GPR30 alters hDF response to 17β-estradiol.

<p><b>a.</b> Expression of the indicated proteins in hDF or MCF7. <b>b.</b> Expression of the indicated proteins upon E2 stimulation. Ratios of phosphorylated ERK (p-ERK) to total ERK (t-ERK) are indicated. <b>c.</b> ERK phosphorylation under the indicated conditions (G-1: GPR30 agonist). Right panel: quantification of the western blots performed on the four donors. Data are presented as mean +/- s.e.m. relative to vehicle-treated control. <b>d.</b> ERK phosphorylation under the indicated conditions (G-15: GPR30 antagonist; PD98059 [PD]: ERK inhibitor). Lower panel: quantification of the results as above. <b>e.</b> Cells shape monitored under the indicated conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120672#pone.0120672.g001" target="_blank">Fig. 1b</a>. n = 400 cells. ns: not significant; *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p