Abstract:

HEK293T cells were triple transfected with PPARγ and RXRα ligand binding domain expression vectors, firefly luciferase reporter vectors and renilla luciferase control vectors. Cells were then exposed to positive and negative control ligands and serial dilutions of dioctyl sodium sulfosuccinate (DOSS) only, water accommodated fraction (WAF) of oil/DOSS mixture, and DOSS water accommodated fraction (CWAF) treatments. Activation of both receptor binding domains was observed in CWAF and DOSS only treatments, exhibiting a dose response curve indicating obesogenic potential of these fractions.

Suggested Citation:

Demetri Spyropoulos, Lexi Temkin, Robert Bowers, John Baatz. 2016. PPARγ and RXRα Ligand Binding Assays. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N798851H

Publications:

Temkin, A. M., Bowers, R. R., Magaletta, M. E., Holshouser, S., Maggi, A., Ciana, P., … Spyropoulos, D. D. (2015). Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen. Environmental Health Perspectives, 124(1). doi:10.1289/ehp.1409672

Bowers, R. R., Temkin, A. M., Guillette, L. J., Baatz, J. E., & Spyropoulos, D. D. (2016). The commonly used nonionic surfactant Span 80 has RXRα transactivation activity, which likely increases the obesogenic potential of oil dispersants and food emulsifiers. General and Comparative Endocrinology, 238, 61–68. doi:10.1016/j.ygcen.2016.04.029

Purpose:

To identify differences in obesogenic potential of DOSS only, WAF, and CWAF treatments for a model human cell line, HEK293

Data Parameters and Units:

Average relative fluorescence intensities of expressed firefly luciferase (PPARgamma-dependent) and renilla luciferase (RXRalpha-dependent) were measured in HEK293 cells as a function of DOSS, WAF or CWAF exposure. PPARg_Ligand Binding Assays_Controls.csv Ligand binding assay controls for normalized luciferase (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol PPARg_Ligand Binding Assays_oil treatment.csv Ligand binding assay - oil treatment for normalized luciferase (firefly luciferase / renilla luciferase ratios) WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control PPARg_RXRa_Ligand Binding Assay_DIF.docx Information about the dataset and dataset file list PPARg_RXRa_Ligand Binding Assays_Controls.pdf Graphs for domain receptor plasmid for control treatments TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol PPARg_RXRa_Ligand Binding Assays_Controls.xlsx Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 PPARg Control Raw: Ligand binding assay controls for luciferase raw and normalized values (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol PPARg Control Normalized: Ligand binding assay controls for normalized luciferase (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol RXRa Control Raw: Ligand binding assay controls for luciferase raw and normalized values (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol RXRa Control Normalized: Ligand binding assay controls for normalized luciferase (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol PPARg_RXRa_Ligand Binding Assays_Oil Treatments.pdf Graphs for domain receptor plasmid for oil treatments WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control PPARg_RXRa_Ligand Binding Assays_Oil Treatments.xlsx Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 PPARg Oil Treatment Raw: Ligand binding assay - oil treatment raw and normalized values (firefly luciferase / renilla luciferase ratios) WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control PPARg Oil Treatment Normalized: Ligand binding assay - oil treatment for normalized luciferase (firefly luciferase / renilla luciferase ratios) WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control RXRa Oil Treatment Raw: Ligand binding assay - oil treatment raw and normalized values (firefly luciferase / renilla luciferase ratios) WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control RXRa Oil Treatment Normalized: Ligand binding assay - oil treatment for normalized luciferase (firefly luciferase / renilla luciferase ratios) WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control RXRa_Ligand Binding Assays_Controls.csv Ligand binding assay controls for normalized luciferase (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol RXRa_Ligand Binding Assays_oil treatment.csv Ligand binding assay - oil treatment for normalized luciferase (firefly luciferase / renilla luciferase ratios) WAF: WAF is MC252 oil and DMEM/F12 mixed 1:10. CWAF: CWAF (COREXIT Water Accommodated Fraction) is COREXIT 9500, MC252 oil and DMEM/F12 cell culture media mixed 1:20:200 (ratios by volume) Corexit: COREXIT only mixtures are COREXIT and DMEM/F12 mixed 1:200 Control

Individual Files: