PPARγ and RXRα Ligand Binding Assays: Corexit Components
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Funded By:
Gulf of Mexico Research Initiative
Funding Cycle:
RFP-II
Research Group:
Using Embryonic Stem Cell Fate to Determine Potential Adverse Effects of Petroleum/Dispersant Exposure
Demetri D. Spyropoulos
Medical University of South Carolina / Marine Biomedicine and Environmental Sciences
spyropdd@musc.edu
Adipogenesis, Corexit, gene expression
Abstract:
HEK293T cells were triple transfected with PPARγ ligand binding domain expression vectors, firefly luciferase reporter vectors and renilla luciferase control vectors. Cells were then exposed to positive and negative control ligands (Rosiglitazone and DMSO respectively) and serial dilutions of DOSS, ICP:PG:DOSS, ICP:PG and Span80 treatments. Activation of the PPARγ binding domain was observed in DOSS containing treatments only, exhibiting a dose response curve indicating obesogenic potential DOSS.
Suggested Citation:
Demetri Spyropoulos, Lexi Temkin, Robert Bowers, John Baatz. 2016. PPARγ and RXRα Ligand Binding Assays: Corexit Components. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N75H7D8B
Publications:
Purpose:
To identify components of Corexit that induce adipogenesis in a human cell line.
Data Parameters and Units:
Average fluorescence intensities of expressed firefly luciferase (PPARgamma dependent) was determined as a function of exposure to Corexit or specific components of Corexit (ICP:PG:DOSS, ICP:PG and Span80). Intensities for cells containing reporter vectors were compared to cells with control expression vectors. DMSO and Rosiglitizone were used as negative and positive controls, respectively. PPARg_Ligand Binding Assay_COREXIT Components_DIF.docx Information about the dataset and dataset file list PPARg_Ligand Binding Assays_COREXIT Components.csv Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 Ligand binding assay - COREXIT for normalized luciferase (firefly luciferase / renilla luciferase ratios) Control DOSS: COREXIT component, dioctyl sodium sulfosuccinate (DOSS) ICP-PG-DOSS: inductively coupled plasma - propylene glycol - dioctyl sodium sulfosuccinate ICP-PG: inductively coupled plasma - propylene glycol SPAN-80 PPARg_Ligand Binding Assays_COREXIT Components.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 COREXIT Comp Raw: Ligand binding assay - COREXIT for raw and normalized luciferase (firefly luciferase / renilla luciferase ratios) Control DOSS: COREXIT component, dioctyl sodium sulfosuccinate (DOSS) ICP-PG-DOSS: inductively coupled plasma - propylene glycol - dioctyl sodium sulfosuccinate ICP-PG: inductively coupled plasma - propylene glycol SPAN-80 PPARg COREXIT Comp Norm: Ligand binding assay - COREXIT for normalized luciferase (firefly luciferase / renilla luciferase ratios) Control DOSS: COREXIT component, dioctyl sodium sulfosuccinate (DOSS) ICP-PG-DOSS: inductively coupled plasma - propylene glycol - dioctyl sodium sulfosuccinate ICP-PG: inductively coupled plasma - propylene glycol SPAN-80 PPARg_Ligand Binding Assays_Controls.csv Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 Ligand binding assay controls for luciferase normalized values (firefly luciferase / renilla luciferase ratios) TBT: Tributyltin Rosi: Rosiglitizone RA: all-trans-retinoic acid E2: 17β-estradiol PPARg_Ligand Binding Assays_oil treatments.csv Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 Ligand binding assay - oil treatment 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_RXRa_Ligand Binding Assay_DIF.docx Information about the dataset and dataset file list 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.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 Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 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 Ratio 1 = Firefly luciferase 1 / Renilla luciferase 1 Ratio 2 = Firefly luciferase 2 / Renilla luciferase 2 Ratio 3 = Firefly luciferase 3 / Renilla luciferase 3 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 TBT: Tributyltin