Overexpression and Knockdown of RAE1 in Breast Cancer Cells

MCF7, T47D, and MDA-MB-231 cells were cultured as previously described27 (link). For overexpression studies, full-length RAE1 cDNA was cloned in a pCMV6 expression vector containing the Myc-DDK-tag (#PS100001; Origene, Rockville, MD, USA); this vector construct was subsequently transfected into MCF7, T47D, and MDA-MB-231 cells using Attractene reagent (Qiagen, Venlo, Netherlands). For controls, cells were transfected with an empty pCMV6 vector. Transfected cells were treated with G418 (500 μg/ml; Gibco, Grand Island, NY, USA) for 2–3 weeks to generate stable cell lines. For knockdown experiments, MCF7 and MDA-MB-231 cells were transduced with pGFP-C-shLenti virus particles specific for RAE1 (#TR30023; Origene) or control shRNA. These cells were treated with puromycin (0.5 μg/ml; Sigma, St. Louis, MO, USA) for 7 days to generate stable cell lines.