FIG. 1. Comparison of luciferase signal in ZFL, ZELH, ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2 cells either left untreated (DMSO control) or exposed to.

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FIG. 1. Comparison of luciferase signal in ZFL, ZELH, ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2 cells either left untreated (DMSO control) or exposed to E2. Relative luminescent units were expressed for 25,000 cells/per well. Results are means of triplicates ± SD; *Statistically different from DMSO control within the cell line (p < 0.05). From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com

FIG. 2. Concentration-dependent luciferase induction in response to 17β-estradiol (E2) in ZFL cells either transiently (solid motifs/dash line) or stably (open motif/plain line) transfected with zfER subtypes and ERE-βGlob-Luc-SV-hygro. Luciferase activity is expressed as percentage of maximal luciferase induction by E2 10nM in ZELH-zfERα cells and E2 3nM in ZELH-zfERβ1 and ZELH-zfERβ2 cells. Results are means of triplicates ± SD and are representative of three (transient) or 14–15 (stable) independent experiments. From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com

FIG. 3. Expression of zfERα, zfERβ1, and zfERβ2 mRNA in stably transfected ZELH-zfERs and parental ZFL cell lines as detected by real-time PCR. Results are expressed as relative levels of zfER mRNA level in stably transfected cells over that measured in parental ZFL cell line. Results (means ± SD) are representative of two independent experiments. From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com

FIG. 4. Effect of E2 on cell proliferation in ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2 cell lines, as determined by MTT coloration test. Results are expressed as % of DMSO-treated cells means ± SD of triplicates and are representative of three independent experiments; *Statistically different from DMSO control (p < 0.05). From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com

FIG. 5. Dose response curves of luciferase induction in ZELH-zfERα by various ER ligands: (A) natural estrogens: 17β-estradiol (E2), estrone (E1), and estriol (E3); industrial chemicals: 4-tert-octylphenol (4tOP), bisphenol A (BPA), and 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p′-DDT) (B) pharmaceutical compounds: diethylstilbestrol (DES), hexestrol (Hex), and 17α-ethynylestradiol (EE2), (C) α-zearalenol (α-Zee), β-zearalenol (β-Zee), α-zearalanol (α-Zea), and genistein (Gen), (D) benzophenone derivates: 2,4-dihydroxybenzophenone (benzophenone 1 or BP1), 2,2’,4,4’-tetrahydroxybenzophenone (benzophenone 2 or BP2), and 2,4,4’-trihydroxybenzophenone (THB). Cells were exposed for 72 h; values are expressed as percentage of luciferase induction by E2 10nM (means ± SD of triplicates). From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com

FIG. 6. Dose response curves of luciferase induction in ZELH-zfERβ1 by various ER ligands: (A) natural estrogens: 17β-estradiol (E2), estrone (E1), and estriol (E3), (B) pharmaceutical compounds: diethylstilbestrol (DES), hexestrol (Hex), and 17α-ethynylestradiol (EE2), (C) α-zearalenol (α-Zee), β-zearalenol (β-Zee), α-zearalanol (α-Zea), and genistein (Gen), (D) benzophenone derivates: 2,4-dihydroxybenzophenone (benzophenone 1 or BP1), 2,2’,4,4’-tetrahydroxybenzophenone (benzophenone 2 or BP2), and 2,4,4’-trihydroxybenzophenone (THB). Cells were exposed for 72 h; values are expressed as percentage of luciferase induction by E2 3nM (means ± SD of triplicates). From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com

FIG. 7. Dose response curves of luciferase induction in ZELH-zfERβ2 by various ER ligands: (A) natural estrogens: 17β-estradiol (E2), estrone (E1), and estriol (E3); industrial chemicals: 4-tert-octylphenol (4tOP), bisphenol A (BPA), and 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p′-DDT) (B) pharmaceutical compounds: diethylstilbestrol (DES), hexestrol (Hex), and 17α-ethynylestradiol (EE2), (C) α-zearalenol (α-Zee), β-zearalenol (β-Zee), α-zearalanol (α-Zea), and genistein (Gen), (D) benzophenone derivates: 2,4-dihydroxybenzophenone (benzophenone 1 or BP1), 2,2’,4,4’-tetrahydroxybenzophenone (benzophenone 2 or BP2), and 2,4,4’-trihydroxybenzophenone (THB). Cells were exposed for 72 h; values are expressed as percentage of luciferase induction by E2 3nM (means ± SD of triplicates). From: Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line Toxicol Sci. 2011;125(2):439-449. doi:10.1093/toxsci/kfr297 Toxicol Sci | © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com