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. 2020 Feb 27;128(2):027008. doi: 10.1289/EHP5862

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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 5A comprises four bar graphs. The first and third bar graphs plot relative proliferation effect, ranging from 0 to 60 in increments of 10 (y-axis) for control, 10 nanomolar BPA, 10 superscript 2 nanomolar BPA, and 10 superscript 3 nanomolar BPA across ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha beta superscript negative virgule negative 2 hash and ER beta superscript positive virgule positive, ER beta superscript negative virgule negative 1 hash, and ER beta superscript negative virgule negative 2 hash, respectively, (x-axis). The second and the fourth graphs plot relative proliferation effect, ranging from 0 to 60 in increments of 10 (y-axis) for control, 10 nanomolar BPS, 10 superscript 2 nanomolar BPS, and 10 superscript 3 nanomolar BPS across ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha beta superscript negative virgule negative 2 hash and ER beta superscript positive virgule positive, ER beta superscript negative virgule negative 1 hash, and ER beta superscript negative virgule negative 2 hash, respectively, (x-axis). Figure 5B comprises two bar graphs. The first and second bar graphs plot 5hmC virgule dC, ranging from 0.00000 to 0.00016 in increments of 0.00002 (y-axis) for control, 100 nanomolar BPA, and 100 nanomolar BPS across ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha beta superscript negative virgule negative 2 hash and ER beta superscript positive virgule positive, ER beta superscript negative virgule negative 1 hash, and ER beta superscript negative virgule negative 2 hash, respectively, (x-axis). Figure 5C comprises two bar graphs. The first and second bar graphs plot relative intensity, ranging from 0 to 35 percent in increments of 5 (y-axis) for control, ER alpha superscript positive virgule positive plus 100 nanomolar BPA, ER alpha superscript negative virgule negative 1 hash plus 100 nanomolar BPA, ER alpha superscript negative virgule negative 2 hash plus 100 nanomolar BPA and control, ER alpha superscript positive virgule positive plus 100 nanomolar BPS, ER alpha superscript negative virgule negative 1 hash plus 100 nanomolar BPS, ER alpha superscript negative virgule negative 2 hash plus 100 nanomolar BPS, respectively, over TET 1, TET2, and TET3 (x-axis). Figure 5D comprises a set of two Western blots and a bar graph. The Western blots display TET2-shRNA positive and negative in ER alpha superscript negative virgule negative 1 hash MCF7 and positive and negative in ER alpha superscript negative virgule negative 2 hash MCF7 across the top as columns and TET2 and GAPDH across the left as rows. The graph plots relative intensity, ranging from 0 to 35 percent in increments of 5 (y-axis) across ER alpha superscript negative virgule negative 1 hash, ER alpha superscript negative virgule negative 1 hash plus shTET2, ER alpha superscript negative virgule negative 2 hash, and ER alpha superscript negative virgule negative 2 hash plus shTET2 (x-axis). Figures 5E and 5F plot relative proliferation effect, ranging from 0 to 60 in increments of 10 and 5hmC virgule dC from 0.00000 to 0.00016 in increments of 0.00002, respectively, (y-axis) for ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha superscript negative virgule negative 2 hash, ER alpha superscript negative virgule negative 1 hash plus shTET2, and ER alpha superscript negative virgule negative 2 hash plus shTET2 across control, 100 nanomolar BPA, and 100 nanomolar BPS (x-axis). — Cell proliferation, 5-hydroxymethylcytosine ( $5 hmC$ ) level, and ten-eleven translocation 2 (TET2) expression in estrogen-receptor-knockout MCF-7 cells treated with bisphenol A (BPA) or bisphenol S (BPS). (A) Cell proliferation assay by MTS of ${ER α}^{- / -}$ and ${ER β}^{- / -}$ MCF-7 cells exposed to vehicle, BPA, or BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical analysis was performed with two-way ANOVA (with Bonferroni posttest): *, $p < 0.05$ or **, $p < 0.01$ vs. control of ${ER α}^{+ / +}$ or ${ER β}^{+ / +}$ cells. (B) $5 hmC$ frequency measured by UHPLC-MRM MS/MS analysis of digested genomic DNA from ${ER α}^{- / -}$ and ${ER β}^{- / -}$ MCF-7 cells exposed to vehicle, BPA, or BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical analysis was performed with two-way ANOVA (with Bonferroni posttest). *, $p < 0.05$ or **, $p < 0.01$ vs. control of ${ER α}^{+ / +}$ or ${ER β}^{+ / +}$ cells. (C) Quantification of Western blot detection of TET1, TET2, and TET3 proteins in ${ER α}^{- / -}$ MCF-7 cells at 48 h after vehicle, BPA, or BPS treatment. The relative intensity was analyzed with ImageJ software (Schneider et al. 2012) and calculated by the ratio relative to the GAPDH intensity. Data represent $mean \pm SD$ of three independent experiments. Statistical analysis was performed with Student’s paired t-test. *, $p < 0.05$ vs. control. (D) Identification of TET2 protein in ${ER α}^{- / -}$ MCF-7 cells transfected with short hairpin RNA (shRNA)-TET2 plasmid using Western blot. The relative intensity was analyzed with ImageJ software and calculated by the ratio relative to the GAPDH intensity. Data represent $mean \pm SD$ of three independent experiments. Statistical analysis was performed with Student’s paired t-test. ^#, $p < 0.05$ vs. indicated samples. (E) Cell proliferation assay by MTS of ${ER α}^{- / -}$ MCF-7 cells transfected with shRNA-TET2 exposed to BPA and BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical analysis was performed with two-way ANOVA (with Bonferroni posttest). *, $p < 0.05$ or **, $p < 0.01$ vs. ${ER α}^{+ / +}$ of each treatment. (F) $5 hmC$ frequency measured by UHPLC-MRM MS/MS analysis of digested genomic DNA from ${ER α}^{- / -}$ MCF-7 cells transfected with shRNA-TET2 exposed to BPA and BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical significance was evaluated by two-way ANOVA (with Bonferroni posttest). *, $p < 0.05$ or **, $p < 0.01$ vs. control of ${ER α}^{+ / +}$ cells. Note: ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MTS, a tetrazolium salt; SD, standard deviation; UHPLC-MRM MS/MS, ultra-high performance liquid chromatography–tandem mass spectrometry.

Figure 5A comprises four bar graphs. The first and third bar graphs plot relative proliferation effect, ranging from 0 to 60 in increments of 10 (y-axis) for control, 10 nanomolar BPA, 10 superscript 2 nanomolar BPA, and 10 superscript 3 nanomolar BPA across ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha beta superscript negative virgule negative 2 hash and ER beta superscript positive virgule positive, ER beta superscript negative virgule negative 1 hash, and ER beta superscript negative virgule negative 2 hash, respectively, (x-axis). The second and the fourth graphs plot relative proliferation effect, ranging from 0 to 60 in increments of 10 (y-axis) for control, 10 nanomolar BPS, 10 superscript 2 nanomolar BPS, and 10 superscript 3 nanomolar BPS across ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha beta superscript negative virgule negative 2 hash and ER beta superscript positive virgule positive, ER beta superscript negative virgule negative 1 hash, and ER beta superscript negative virgule negative 2 hash, respectively, (x-axis). Figure 5B comprises two bar graphs. The first and second bar graphs plot 5hmC virgule dC, ranging from 0.00000 to 0.00016 in increments of 0.00002 (y-axis) for control, 100 nanomolar BPA, and 100 nanomolar BPS across ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha beta superscript negative virgule negative 2 hash and ER beta superscript positive virgule positive, ER beta superscript negative virgule negative 1 hash, and ER beta superscript negative virgule negative 2 hash, respectively, (x-axis). Figure 5C comprises two bar graphs. The first and second bar graphs plot relative intensity, ranging from 0 to 35 percent in increments of 5 (y-axis) for control, ER alpha superscript positive virgule positive plus 100 nanomolar BPA, ER alpha superscript negative virgule negative 1 hash plus 100 nanomolar BPA, ER alpha superscript negative virgule negative 2 hash plus 100 nanomolar BPA and control, ER alpha superscript positive virgule positive plus 100 nanomolar BPS, ER alpha superscript negative virgule negative 1 hash plus 100 nanomolar BPS, ER alpha superscript negative virgule negative 2 hash plus 100 nanomolar BPS, respectively, over TET 1, TET2, and TET3 (x-axis). Figure 5D comprises a set of two Western blots and a bar graph. The Western blots display TET2-shRNA positive and negative in ER alpha superscript negative virgule negative 1 hash MCF7 and positive and negative in ER alpha superscript negative virgule negative 2 hash MCF7 across the top as columns and TET2 and GAPDH across the left as rows. The graph plots relative intensity, ranging from 0 to 35 percent in increments of 5 (y-axis) across ER alpha superscript negative virgule negative 1 hash, ER alpha superscript negative virgule negative 1 hash plus shTET2, ER alpha superscript negative virgule negative 2 hash, and ER alpha superscript negative virgule negative 2 hash plus shTET2 (x-axis). Figures 5E and 5F plot relative proliferation effect, ranging from 0 to 60 in increments of 10 and 5hmC virgule dC from 0.00000 to 0.00016 in increments of 0.00002, respectively, (y-axis) for ER alpha superscript positive virgule positive, ER alpha superscript negative virgule negative 1 hash, ER alpha superscript negative virgule negative 2 hash, ER alpha superscript negative virgule negative 1 hash plus shTET2, and ER alpha superscript negative virgule negative 2 hash plus shTET2 across control, 100 nanomolar BPA, and 100 nanomolar BPS (x-axis). — Cell proliferation, 5-hydroxymethylcytosine ( $5 hmC$ ) level, and ten-eleven translocation 2 (TET2) expression in estrogen-receptor-knockout MCF-7 cells treated with bisphenol A (BPA) or bisphenol S (BPS). (A) Cell proliferation assay by MTS of ${ER α}^{- / -}$ and ${ER β}^{- / -}$ MCF-7 cells exposed to vehicle, BPA, or BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical analysis was performed with two-way ANOVA (with Bonferroni posttest): *, $p < 0.05$ or **, $p < 0.01$ vs. control of ${ER α}^{+ / +}$ or ${ER β}^{+ / +}$ cells. (B) $5 hmC$ frequency measured by UHPLC-MRM MS/MS analysis of digested genomic DNA from ${ER α}^{- / -}$ and ${ER β}^{- / -}$ MCF-7 cells exposed to vehicle, BPA, or BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical analysis was performed with two-way ANOVA (with Bonferroni posttest). *, $p < 0.05$ or **, $p < 0.01$ vs. control of ${ER α}^{+ / +}$ or ${ER β}^{+ / +}$ cells. (C) Quantification of Western blot detection of TET1, TET2, and TET3 proteins in ${ER α}^{- / -}$ MCF-7 cells at 48 h after vehicle, BPA, or BPS treatment. The relative intensity was analyzed with ImageJ software (Schneider et al. 2012) and calculated by the ratio relative to the GAPDH intensity. Data represent $mean \pm SD$ of three independent experiments. Statistical analysis was performed with Student’s paired t-test. *, $p < 0.05$ vs. control. (D) Identification of TET2 protein in ${ER α}^{- / -}$ MCF-7 cells transfected with short hairpin RNA (shRNA)-TET2 plasmid using Western blot. The relative intensity was analyzed with ImageJ software and calculated by the ratio relative to the GAPDH intensity. Data represent $mean \pm SD$ of three independent experiments. Statistical analysis was performed with Student’s paired t-test. ^#, $p < 0.05$ vs. indicated samples. (E) Cell proliferation assay by MTS of ${ER α}^{- / -}$ MCF-7 cells transfected with shRNA-TET2 exposed to BPA and BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical analysis was performed with two-way ANOVA (with Bonferroni posttest). *, $p < 0.05$ or **, $p < 0.01$ vs. ${ER α}^{+ / +}$ of each treatment. (F) $5 hmC$ frequency measured by UHPLC-MRM MS/MS analysis of digested genomic DNA from ${ER α}^{- / -}$ MCF-7 cells transfected with shRNA-TET2 exposed to BPA and BPS. Data represent $mean \pm SD$ of five independent experiments. Statistical significance was evaluated by two-way ANOVA (with Bonferroni posttest). *, $p < 0.05$ or **, $p < 0.01$ vs. control of ${ER α}^{+ / +}$ cells. Note: ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MTS, a tetrazolium salt; SD, standard deviation; UHPLC-MRM MS/MS, ultra-high performance liquid chromatography–tandem mass spectrometry.