Dextran coated charcoal stripped serum

COS-7 cells were seeded in phenol red-free DMEM (Wisent) supplemented with 5% dextran-coated charcoal stripped serum (Sigma-Aldrich). Twenty-four hours after plating, cells were transfected with plasmid DNA using Fugene HD (Promega) according to the manufacturer’s recommendations. For the transcriptional assays, cells were transfected with 10 ng of pRL-CMV (renilla; internal control, Promega) 25 ng of VP-16-ERα 25 ng of VP-16-ERβ (Addgene^{38 (link)} and 125 ng of 3X ERE-TATA-luciferase (ERE-luc) (Addgene^{38 (link)}. Twenty-four hours after transfection, cells were treated with vehicle control and the indicated concentrations of BPS or BPA, as well as 1 nM E2 as positive control. Twenty-four after treatment, cells were lysed using 1X Passive Lysis Buffer (Promega). Luciferase activity was quantified with the Dual Luciferase Assay kit (Promega) using the Glomax96 Luminometer (Promega). Luciferase activity was normalized to Renilla levels and to vehicle control.

COS-7 cells were seeded in phenol red-free DMEM (Wisent) supplemented with 5% dextran-coated charcoal stripped serum (Sigma-Aldrich). Twenty-four hours after plating, cells were transfected with plasmid DNA using Fugene HD (Promega) according to the manufacturer’s recommendations. For the PPARγ transcriptional assays, cells were transfected with 10 ng of pRL-CMV (renilla; internal control), 25 ng of pcDNA mPPARγ, 25 ng of pCMV6 mRXR, and 125 ng of aP2 enhancer-luciferase (aP2 enhancer-luc). Murine aP2-luciferase was a gift from Bruce Spiegelman (Addgene plasmid #8858). This 520 bp enhancer construct is 5.1 kb upstream from the transcriptional start site and contains a functional PPRE (PPARγ response element). Six hours after transfection, cells were treated with vehicle control and the indicated concentrations of TROG, FM550, TPP, IPTP, DPP, TBB, or TBPH. Twenty-four after treatment, cells were lysed using 1X Passive Lysis Buffer (Promega). Luciferase activity was quantified with the Dual Luciferase Assay kit (Promega) using the Glomax96 Luminometer (Promega). Luciferase activity was normalized to renilla levels and to vehicle control (DMSO).