Pmission vsv g

All cell lines were authenticated in 2014 by Short Tandem Repeat DNA Profiling (Promega; Fitchburg, WI) at the University of Colorado Cancer Center (UCCC) Tissue Culture Core, and testing for mycoplasma was performed every three months. Only cells under five passages were used in this study. SUM159PT cells were purchased from the UCCC Tissue Culture Core in 2013 and were grown in Ham’s F-12 with 5% fetal bovine serum (FBS), penicillin/streptomycin (P/S), hydrocortisone, insulin, HEPES and L-glutamine supplementation. BT549 cells, purchased from the ATCC in 2008, were grown in RPMI Medium 1640 with 10% FBS, P/S and insulin. MDA-MB-453 (MDA453) cells, purchased from ATCC in 2012, were grown in DMEM Medium with 10% FBS and P/S.
SUM159PT-TGL cells were generated by stable retroviral transduction with a SFG-NES-TGL vector, encoding a triple fusion of thymidine kinase, green fluorescent protein and luciferase and sorted for green fluorescent protein. SUM159PT and MDA453 AR knockdown cells were generated by lentiviral transduction of shRNAs targeting AR (pMISSION VSV-G, Sigma Aldrich; St Louis, MO), including AR shRNA 3715 (shAR15) and AR shRNA 3717 (shAR17). Lentiviral transduction of pMISSION shRNA NEG (shNEG) was used as a non-targeting control. Plasmids were purchased from the University of Colorado Functional Genomics Core Facility.

Lentiviral shRNA vectors (pLKO.1) were either purchased from Sigma or assembled by cloning the shRNA hairpin loop sequence into the AgeI/EcoRI sites of the empty vector. The target sequence for the ZRSR2 shRNAs sh1 and sh2 are 5’-CAACAGTTCCTAGACTTCTAT-3’ and 5’-AGCAGCCCTTTCTCTGTTTAA-3’, respectively. MISSION pLKO.1-puro Non-Mammalian shRNA Control (SHC002; Sigma) was used as control vector for transduction. To generate lentiviral particles, 293T cells (kindly provided by Dr. Bing Lim, Genome Institute of Singapore, Singapore) were co-transfected with shRNA plasmid and the packaging plasmids, pMISSIONgagpol and pMISSIONvsvg (Sigma) using Lipofectamine 2000 (Invitrogen). Virus containing supernatant was collected after 48 and 72 hours, filtered through 0.45 μm filter and stored in aliquots at −80°C. TF-1 and K562 cells (American Type Culture Collection) were infected with lentivirus for 2 rounds, 24 hours apart, in the presence of 5 μg/ml protamine sulfate. Transduced cells were selected in puromycin to generate stable knockdown cell lines. The knockdown was verified using qPCR and western blotting.