Glomax 96 luminometer

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.

MCF-7 cells (7 × 10⁴) were seeded in 500 µL DMEM with FBS in 24-well plates and grown for 48 hours. Cells were co-transfected with 0.5 µg of each luciferase construct with or without 10 nM hsa-miR-494-3p miRNA mimic or Negative Control #1 using 1.5 µL Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions. The cells were in addition co-transfected with pRL-SV40 vector (5 ng) expressing Renilla luciferase DNA (hRluc). Medium was replaced four hours post transfection. After 48 hours, cells were washed in Phosphate-buffered saline (PBS) (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and lysed with 100 µL of passive lysis buffer (1×), before the luciferase activity was quantified in a Glomax®−96 Luminometer (Promega) using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Values were corrected for Renilla internal control activity. The luciferase activity from unstimulated cells transfected with the pGL3-promoter was arbitrarily defined as 1 and the miR transfected cells were adjusted accordingly.