Transient transfection of the cell lines and progestin administration

To investigate the roles of Nrf2 and AKR1C1 in progestin resistance, exogenous transfection of Nrf2 and AKR1C1 were performed. Briefly, after serum starvation for 24 hours, Ishikawa and RL95–2 cells were transfected with pCI-Nrf2 or pCI-AKR1C1 expression plasmids using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The transfection efficiency was determined by Western blot analysis. After transfection for 16 h, Nrf2- or AKR1C1-transfected cells and their corresponding controls were treated with different dose of MPA for another 48 hours. Cell proliferation was determined by the MTT assay.