br CONTACT FOR REAGENT AND RESOURCE
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be direct to and will be fulfilled by the Lead Contact, Myles Brown ([email protected]).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
LNCaP95 Oxidopamine hydrochloride have been previously described (Hu et al., 2012) and were maintained in phenol red-free RPMI 1640 medium, sup-plemented with 10% charcoal/dextran-treated fetal bovine serum (FBS), 1% L-Glutamine and 1% penicillin/streptomycin. 22Rv1 cells were obtained from the American Type Culture Collection (ATCC) and were grown in regular RPMI 1640 medium, supplemented with 10% FBS, 1% L-Glutamine and 1% penicillin/streptomycin. For experiments requiring hormone starvation, 22Rv1 cells were grown in phenol red-free RPMI 1640 medium supplemented with 10% charcoal/dextran-treated FBS, 1% L-Glutamine and 1% peni-cillin/streptomycin for 72 hr prior to any subsequent vehicle or hormone addition. All cell lines were tested for mycoplasma using the MycoAlert mycoplasma detection kit (Lonza, Portsmouth, NH) and their identities were confirmed by short tandem repeat profiling (BioSynthesis, Lewisville, TX).
Clinical Patient Samples
A set of 59 metastatic tumors from 37 men with CRPC were obtained with informed consent through the University of Washington Prostate Cancer Donor Autopsy Program (Morrissey et al., 2013) and used for transcript profiling by microarray, as described (Kumar et al., 2016) using frozen tissues. Soft tissue tumors were laser capture-microdissected and bone metastases were sampled using a 1 mm diameter tissue punch. All procedures involving human subjects were approved by the Institutional Review Board (IRB) of the University of Washington and of the Fred Hutchinson Cancer Research Center.
Generation of Doxycycline-Inducible shARfl or shARv7 Cell Lines
Doxycycline (dox)-inducible cell lines were generated using lentiviral vectors in pLKO-Tet-On backbones (Wiederschain et al., 2009) targeting GFP, ARfl (exon 8) or ARv7 (cryptic exon 3). Oligonucleotides containing the shRNA sequences, and AgeI and EcoRI restric-tion enzyme sites compatible with cloning into the pLKO-Tet-On vector, were designed using following primers (shRNA sequences are shown in bold): shGFP forward:
5’-CCGGGCAAGCTGACCCTGAAGTTCACTCGAGTGAACTTCAGGGTCAGCTTGCTTTTTG-3’ shGFP reverse:
5’-AATTCAAAAAGCAAGCTGACCCTGAAGTTCACTCGAGTGAACTTCAGGGTCAGCTTGC-3’ shARfl forward:
5’-CCGGCCTGCTAATCAAGTCACACATCTCGAGATGTGTGACTTGATTAGCAGGTTTTTG-3’ shARfl reverse:
5’-AATTCAAAAACCTGCTAATCAAGTCACACATCTCGAGATGTGTGACTTGATTAGCAGG-3’ shARv7 forward:
Oligonucleotides were annealed in 1x annealing buffer (1 M NaCl, 100 mM Tris-HCl, pH 7.4) and heated for 5 min at 95 C. Annealed oligos were ligated into pre-digested (EcoRI and AgeI) pLKO-Tet-On lentiviral plasmid and subsequently transformed into Stbl3 competent bacteria. Lentiviral particles were generated using calcium phosphate, and transfer, VSV-G envelope (pMD2G, Addgene #12259) and packaging vectors (pCMVR8.74, Addgene #22036) (Barde et al., 2010). Prostate cancer cells were infected with uncon-centrated virus and selected with 1 mg/ml puromycin. shRNA expression was induced by treating cells with 1 mg/ml dox for at least 72 hr.
ON-TARGETplus siRNA for non-targeting siCtrl, siNCOR1, siNCOR2, and siNRIP1 was purchased from GE Dharmacon.
Name Catalog Sequence
Transfection of siRNA was performed using Lipofectamine RNAiMax (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Cell Proliferation Assays
For the proliferation assays, indicated cell lines were plated in a 24-well plate format at 2x104 cells/well. Unless otherwise stated, cells were induced for 3 days with dox prior to cell growth assessment. Cell growth was determined for indicated times by trypan blue exclusion using a hemocytometer, or by using the direct cell count function on a Celigo Imaging Cytometer (Nexcelom Bioscience). 3D cell proliferation assay was performed using poly(ethylene glycol) diacrylate 575 cryogels (Go¨ppert et al., 2016), incubated with 2.5 x 105 cells in a 12-well plate format. Dox was added one day after seeding. Cryogels were removed at indicated time points and cells lysed using 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulfate and 200 mg/ml Proteinase K (Sigma-Aldrich). DNA was extracted and quantified and cell growth visualization was carried out using scanning electron microscopy.