For transfection (1.5 × 105), CHO-K1 cells were plated in each well of a 24-well plate in IMDM without phenol red supplemented with 5% charcoal-stripped calf serum (CSCS) and Pen/Strep. 24 h after plating, the cells were co-transfected with 250 ng reporter (ERE-luciferase) and 5 ng of pRL-CMV (Promega, Madison, WI) as previously described (Thomas et al., 2003 (link)) in IMDM without serum. The cells were treated with the vehicle (DMSO), 100 nM 4-OHT, or 1 μM ICI 128,780. Treatments were performed in triplicate within each experiment. After 24 h of treatment, the cells were lysed in reporter lysis buffer (Promega) and the cleared cell extract was used for dual luciferase reporter assay (Promega) (Thomas et al., 2003 (link)). Statistical evaluation of the data was performed using GraphPad Prism Software (Graph Pad Software, San Diego, CA).
ERα regulation of miR-29b-1/a promoter was determined by plating CHO-K1 cells in triplicate in 24 well plates. Next day, cells were transfected with pGL3 −1530/þ165 miR-29b-1/miR-29a promoter fragment (Mott et al., 2010 (link)) and pGL4-hRluc-TK (Renilla, Promega) using FuGENE HD (Roche) according to manufacturer's protocol. 24 h post transfection, cells were treated for another 24 h prior to performing dual luciferase assay (Promega). Relative expression was determined by dividing averaged values from vehicle control (DMSO) values.
ERα regulation of miR-29b-1/a promoter was determined by plating CHO-K1 cells in triplicate in 24 well plates. Next day, cells were transfected with pGL3 −1530/þ165 miR-29b-1/miR-29a promoter fragment (Mott et al., 2010 (link)) and pGL4-hRluc-TK (Renilla, Promega) using FuGENE HD (Roche) according to manufacturer's protocol. 24 h post transfection, cells were treated for another 24 h prior to performing dual luciferase assay (Promega). Relative expression was determined by dividing averaged values from vehicle control (DMSO) values.