3.2. siRNA Knockdown

siRNA treatments were conducted using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) and Luciferase control, H1.2/H1.4 and H1.4 siRNA according to manufacturer’s protocol. The cells were seeded the day prior to transfection to achieve 50–60% confluency at the time of treatment. siRNA and lipofectamine were diluted in Opti-MEM (Gibco, Gaithesberg, MD, USA), mixed, incubated for 5 min, and the complexes were then added directly to the cells now growing in phenol-red free CD-FBS Media to allow estradiol depletion along with siRNA knockdown simultaneously. After 72 h, the cells were placed in E2/ethanol for 30 min before harvesting.