Sequence of primers for real-time PCR.

<p>Sequence of primers for real-time PCR.</p

Dose-response analysis of 3D cultures of MCF-12A cells grown in Matrigel for 16 days in the presence of E2.

<p>Cells were either incubated with solvent (ethanol 0.5%) or with increasing concentrations of E2. Resulting samples were treated with antibodies against laminin V (red), activated caspase-3 (green) and nuclei visualized with To-PRO 3 (blue), and subsequently analysed by confocal microscopy. Images are representative of each one of the treatments and 3 independent experiments.</p

Impact of ER and GPER inhibitors on acini disruption by estrogens.

<p>Cells were treated with the test compounds (E2, 1 nM; BPA, 10 µM; propylparaben, 10 µM) alone or in combination with ICI 182,780 (1 µM), G-15 (10 nM) or ICI 182,780 (1 µM) + G-15 (10 nM) for 16 days. <b>A</b> Resulting acinar structures were immunostained as described before. Red shows laminin V and basement membrane deposition, green highlights apoptotic cells by staining activated caspase-3 and blue shows cell nuclei (TO-PRO 3 counterstain). Images were visualized by confocal microscopy and represent 3 experiments, run independently. <b>B</b>. Quantification of acinar size following treatment with estrogenic chemicals and inhibitors. White bars represent treatment with individual compounds. Hatched white bars show the effect of the chemicals in the presence of ICI 182,780 and grey bars of test compounds in combination with G-15. Grey hatched bars show the effect of the test chemicals when combined with ICI 182,780 + G-15. <b>C</b>. Number of cells per acini. <b>D</b>. Ratio cell number/acini size calculated for the samples shown in B and C. For all graphs, data results from three independent experiments (10 acini/experiment) and indicates mean ± SEM. * (p<0.05) and ** (p<0.01) highlight significant differences between treatments with estrogenic agents tested individually and treatments with estrogens combined with inhibitors.</p

Confocal images of 3D cultures of epithelial breast MCF-10A cells grown in Matrigel for 16 days.

<p>Samples were either treated with solvent (ethanol 0.5%) or with 10 nM E2. Obtained structures were stained with antibody against laminin V (red) to stain the basement membrane, antibody against activated caspase-3 (green) to identify apoptotic cells and counterstained with To-PRO 3 to visualize cell nuclei (blue). Cross-sections of solvent control structures show a well delineated spheroid with a layer of viable cells in contact with the basement membrane. Two apoptotic cells are seen in the centre of the acini. <b>A</b> is representative of structures that were unaffected by estrogen treatment, as formed structures resemble those obtained in controls. <b>B</b> depicts acini that showed mild disruption after E2 treatment. Images are representative of 3 independent experiments.</p

Quantification of acini size and cell number/acini.

<p>The size of acini (µm) and the number of cells per acini were determined by analyzing confocal images of the incubation periods of 8, 12 and 16 days. Data corresponds to mean ± SEM and results from three independent experiments, where a minimum of 10 representative acini were analysed per experiment. * (p&lt;0.05), ** (p&lt;0.01) and *** (p&lt;0.001) indicate significant differences between treatments and controls.</p

Quantification of the percentage of apoptotic cells per acini.

<p>The percentage of apoptotic cells was calculated for 8, 12 and 16 days incubation periods. This was determined as the number of cells positive to activated caspase-3 (stained green) over the total number of cells in each acini. Data corresponds to mean ± SEM from three independent experiments (10 acini/experiment were analysed). * (p&lt;0.05) and ** (p&lt;0.01) denote significant differences between treatment and controls at the corresponding incubation periods.</p

Time-course analysis of the formation of MCF-12A acini cultured in matrigel in the presence of the test chemicals.

<p>Cells were either treated with solvent (ethanol 0.5%) or with 1 nM E2, 10 µM BPA or 10 µM propylparaben and allowed to grow for 4, 8, 12 and 16 days. The resulting structures were immunostained with antiserum to laminin V (red) to delineate the secretion of basement membrane, activated caspase-3(green) to detect apoptotic cells and counterstained with TO-PRO 3 to visualize the cell nuclei. Confocal images are representative of 3 independent experiments.</p

Receptor status and activation of ERα, ERβ and GPER in MCF-12A and MCF-10A cells.

<p><b>A</b>. ERα, ERβ and GPER expression detected by immunoblotting. <b>B</b>. Regulation of ER-target genes. White bars represent treatment with 1 nM E2. Hatched white bars show the effects of E2 in combination with ICI 182,780 (1 µM). Horizontal dashed line corresponds to the negative controls (0.5% ethanol), set to 1. Data are from 3 independent experiments and error bars are mean ± SEM. **a) shows statistically significant differences (p&lt;0.01) between treatments and controls. **b) (p&lt;0.01) and *b) (p&lt;0.05) highlight significant differences between treatments with E2 tested individually and treatments with E2 combined with ICI 182,780.</p

Concentration-response curves for the 21 tested estrogenic chemicals with regression lines derived from the best fitting models for E-Screen <i>in vitro</i> data.

<p>All agents were tested in at least four independent experiments, run on up to three micro-titer plates, with each plate containing eight increasing concentrations of the test chemical in duplicates (data not shown).</p

Estrogenicity of individual compounds and mixture.

<p>EC10: concentration associated with 10% proliferation rate. Values in brackets denote the upper and lower limits of the approximate 95% confidence interval based on bootstrap replicates; the column “RM” indicates the mathematical regression function, used for describing the concentration response relationships (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088808#pone.0088808-Scholze1" target="_blank">[<i>17</i>]</a> for more details). estimated model parameters, if marked by *, then held fixed, i.e. not estimated.</p