Reporter assays

Estrogen-response-element (ERE) and hormone-response-element (HRE) reporters were assembled by cloning 4X repeats of canonical ERE or HRE sequences into pLSG_LR by Switchgear Genomics (Supplemental File 4). For reporter assays, MM134 cells were hormone-deprived prior to siRNA transfection. 24hrs post-siRNA transfection, cells were transfected with ERE reporter, HRE reporter, RPL10 reporter (housekeeper; Switchgear #S708908), or CMV-Renilla (housekeeper) using LTX/Plus (ThermoFisher) per manufacturer’s instructions. 24hrs post-reporter transfection, cells were treated with vehicle (0.01% EtOH) or 100pM E2 for 24hrs. Reporter activity was assessed by LightSwitch Luciferase Assay (SwitchGear Genomics) per manufacturer’s instructions on a Biotek Neo2 plate reader. Relative luminescence units (RLU) are ERE/HRE output normalized to mean CMV/RPL10 output from matched control wells.