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. 2018 Jan 31;126(1):017012. doi: 10.1289/EHP2505

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EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted.

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Figure 1A shows chemical structures of 17 beta estradiol; ICI 182,780; Bisphenol A, Bisphenol AF, and Bisphenol S. Figure 1B comprises two plots respectively showing activation in Ishikawa cells stably expressing ER alpha cells and vector cells, plotting relative luc activity (fold change) (y-axis) across concentration (log[nanomolar]) (x-axis) for E2, BPA, BPAF, and BPS. Figures 1C and 1D plot the same for two plots showing activation of endogenous ER MCF-7 and BG-1 FR cells and HepG2 cells transfected with ER alpha and ER beta, respectively. Figure 1E comprises three bar graphs with standard error of the mean respectively plotting GREB1, PGR, and WISP-2/CNN5 (y-axis) treated with control, E2 10, BPA 100, BPAF 100, and BPS 100 (x-axis) for negative ICI and positive ICI. Figure 1F comprises two parts. The first part presents two bar graphs with standard error of the mean respectively plotting relative recruitment (percentage of input) (y-axis) treated with control and E2 10 (x-axis) for IgG and ER alpha under amplicon negative 699 virgule negative 422 (AP 1 plus ERE) and amplicon negative 593 virgule negative 422 (ERE). The second part presents the same data respectively plotting relative recruitment (percentage of input) (y-axis) treated with control, BPA 100, BPA 1000, BPAF 100, BPAF 1000, BPAS 100, and BPAS 1000 (x-axis). — Bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS) activate estrogen receptor (ER) estrogen response element (ERE)–mediated transcriptional activity, alter expression of ER target genes, and directly bind to ERE sites in $ER α$ target genes. (A) The chemical structures of the compounds tested in this study. (B) Activation in Ishikawa cells stably expressing $ER α$ cells. (C) Activation of endogenous ER MCF-7 and BG-1 FR cells. (D). Co-transfection with $ER α$ or $ER β$ in HepG2 cells. Cells were transfected with 3xERE-luc and pRL-TK plasmids in MCF-7, BG-1 FR, or co-transfection with the $ER α$ or $ER β$ expression plasmid in HepG2 cells overnight. After changing to fresh starved medium, cells were treated with vehicle control, $1 - 1,000 nM$ estradiol (E2), BPA, BPAF, and BPS for 18 h. The ER ERE-mediated activity was detected by the luciferase reporter assay as described in the materials and methods. Data shown are representative of triplicates as fold increases that were calculated relative to the vehicle $(control) \pm standard error of the mean (SEM)$ , *** $p < 0.001$ , ** $p < 0.01$ , * $p < 0.05$ compared to vehicle control. (E) Changes of the ER target genes in MCF-7 cells. Cells were treated with vehicle control, $10 nM$ E2, or $100 nM$ BPA, BPAF, and BPS, with or without $10 μ M$ ICI 182,780 (ICI) for 18 h, respectively. Total RNA was extracted and used as a template for cDNA synthesis. Gene expression was measured by quantitative PCR (qPCR). Experiments were repeated three times, and results are presented as $mean \pm SEM$ , ** $p < 0.01$ , *** $p < 0.001$ , **** $p < 0.0001$ . (F) BPA-, BPAF-, and BPS-induced $ER α$ binding at an ERE site of the human WISP-2 gene promoter. Cells were treated with vehicle control, $10 nM$ E2, and 100 or $1,000 nM$ BPA, BPAF, or BPS for 40 min. ChIP assay was carried out with $anti - ER α$ antibody using a ChIP Assay Kit as described in the materials and methods. Purified chromatin DNA was used for qPCR analysis by the special ChIP primers for the amplicons $- 699 / - 422$ ( $AP - 1 + ERE$ ) or $- 593 / - 422$ (ERE). Relative recruitments of $ER α$ were normalized to signals obtained from input DNA. Experiments were repeated three times, and results are presented as $mean \pm SEM$ , ** $p < 0.01$ , *** $p < 0.001$ , **** $p < 0.0001$ .

Figure 1A shows chemical structures of 17 beta estradiol; ICI 182,780; Bisphenol A, Bisphenol AF, and Bisphenol S. Figure 1B comprises two plots respectively showing activation in Ishikawa cells stably expressing ER alpha cells and vector cells, plotting relative luc activity (fold change) (y-axis) across concentration (log[nanomolar]) (x-axis) for E2, BPA, BPAF, and BPS. Figures 1C and 1D plot the same for two plots showing activation of endogenous ER MCF-7 and BG-1 FR cells and HepG2 cells transfected with ER alpha and ER beta, respectively. Figure 1E comprises three bar graphs with standard error of the mean respectively plotting GREB1, PGR, and WISP-2/CNN5 (y-axis) treated with control, E2 10, BPA 100, BPAF 100, and BPS 100 (x-axis) for negative ICI and positive ICI. Figure 1F comprises two parts. The first part presents two bar graphs with standard error of the mean respectively plotting relative recruitment (percentage of input) (y-axis) treated with control and E2 10 (x-axis) for IgG and ER alpha under amplicon negative 699 virgule negative 422 (AP 1 plus ERE) and amplicon negative 593 virgule negative 422 (ERE). The second part presents the same data respectively plotting relative recruitment (percentage of input) (y-axis) treated with control, BPA 100, BPA 1000, BPAF 100, BPAF 1000, BPAS 100, and BPAS 1000 (x-axis). — Bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS) activate estrogen receptor (ER) estrogen response element (ERE)–mediated transcriptional activity, alter expression of ER target genes, and directly bind to ERE sites in $ER α$ target genes. (A) The chemical structures of the compounds tested in this study. (B) Activation in Ishikawa cells stably expressing $ER α$ cells. (C) Activation of endogenous ER MCF-7 and BG-1 FR cells. (D). Co-transfection with $ER α$ or $ER β$ in HepG2 cells. Cells were transfected with 3xERE-luc and pRL-TK plasmids in MCF-7, BG-1 FR, or co-transfection with the $ER α$ or $ER β$ expression plasmid in HepG2 cells overnight. After changing to fresh starved medium, cells were treated with vehicle control, $1 - 1,000 nM$ estradiol (E2), BPA, BPAF, and BPS for 18 h. The ER ERE-mediated activity was detected by the luciferase reporter assay as described in the materials and methods. Data shown are representative of triplicates as fold increases that were calculated relative to the vehicle $(control) \pm standard error of the mean (SEM)$ , *** $p < 0.001$ , ** $p < 0.01$ , * $p < 0.05$ compared to vehicle control. (E) Changes of the ER target genes in MCF-7 cells. Cells were treated with vehicle control, $10 nM$ E2, or $100 nM$ BPA, BPAF, and BPS, with or without $10 μ M$ ICI 182,780 (ICI) for 18 h, respectively. Total RNA was extracted and used as a template for cDNA synthesis. Gene expression was measured by quantitative PCR (qPCR). Experiments were repeated three times, and results are presented as $mean \pm SEM$ , ** $p < 0.01$ , *** $p < 0.001$ , **** $p < 0.0001$ . (F) BPA-, BPAF-, and BPS-induced $ER α$ binding at an ERE site of the human WISP-2 gene promoter. Cells were treated with vehicle control, $10 nM$ E2, and 100 or $1,000 nM$ BPA, BPAF, or BPS for 40 min. ChIP assay was carried out with $anti - ER α$ antibody using a ChIP Assay Kit as described in the materials and methods. Purified chromatin DNA was used for qPCR analysis by the special ChIP primers for the amplicons $- 699 / - 422$ ( $AP - 1 + ERE$ ) or $- 593 / - 422$ (ERE). Relative recruitments of $ER α$ were normalized to signals obtained from input DNA. Experiments were repeated three times, and results are presented as $mean \pm SEM$ , ** $p < 0.01$ , *** $p < 0.001$ , **** $p < 0.0001$ .