br Clinical Patient Samples br A set of metastatic tumors
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 Gemcitabine 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.
Tissue microarray was performed on whole human genome Agilent microarrays at the University of Washington (TMA 55). IHC was performed using the rabbit ARv7 monoclonal antibody clone RM7 (RevMab). Antigen retrieval was achieved by microwaving slides in citrate buffer (pH 6.0) for 18 min at 800 W. Endogenous peroxidase was blocked using 3% H2O2 solution. Blocking was performed using the protein block solution from the Novolink polymer detection system (Leica, Wetzlar, Germany). EP343 was diluted 1:200 and the tissue was incubated for 1 hr. The reaction was visualized using the Novolink polymer and DAB chromogen. Nuclear ARv7 on TMA slides were scanned with an Aperio ScanScope AT2 (Leica Biosystems Pathology Imaging, Vista, CA) at 40x (0.25 microns/ pixel), and stored on a server running Aperio eSlide Manager digital slide repository and database software.