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  • br Tissue Microarray br Tissue microarray was performed

    2020-08-12


    Tissue Microarray
    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.
    Protein Assays
    Proteins for Western blotting were isolated using TIVE lysis buffer (50 mM Tris-HCl pH 7.8, 2 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitors), or lysis buffer A (10 mM HEPES pH 7.5, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, protease inhibitors) plus 0.5% NP-40 and lysis buffer C (20 mM HEPES pH 7.5, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Glycerol, 1 mM DTT, protease inhibitors) for total, cytoplasmic and nuclear proteins respectively. Co-immunoprecipitation (co-IP) was carried using the nuclear protein fraction, similarly to previously described (Jehle et al., 2014) and ARv7 antibody-coupled Protein A Dynabeads (Thermo Fisher Scientific). Co-IP experiments with formaldehyde crosslinked material was carried out using isolated chromatin sam-ples, as described below. Western blotting was carried out using following antibodies: AR (N20 and 441, Santa-Cruz Biotechnol-ogies; PG21, Millipore), AR C-terminus (SP242, Spring Bioscience; C-19, Santa-Cruz Biotechnologies), ARv7 (RM7, RevMAb; H6 253, Epitomics), NCOR1 (PA1-844A, Invitrogen), NCOR2 (ab24551, Abcam), FOXA1 (ab5089, Abcam), b-Actin (Abcam) and Lamin B1 (EPR8985, Abcam), and secondary antibody IgG fraction monoclonal mouse anti-rabbit IgG, light chain specific (211-032-171, Jackson ImmunoResearch).
    RNA-Sequencing (RNA-seq)
    Unless otherwise stated, 1971007-91-6 were induced for 3 days with dox and treated with vehicle (ETOH) or DHT for 4 hr prior to RNA isolation. Total genomic RNA was isolated using TRIzol (Thermo Fisher Scientific) and the RNeasy Mini Kit (Qiagen) following the manufac-turer’s instructions. mRNA libraries were generated by the Center for Functional Cancer Epigenetics (Dana-Farber Cancer Institute (DFCI)) using 1 mg of total RNA and the Illumina TruSeq stranded mRNA sample kit. Libraries were sequenced on the Illumina NextSeq 500 platform at the Molecular Biology Core Facility (DFCI).
    ChIP-Sequencing (ChIP-seq)
    Unless otherwise stated, cells were induced for 3 days with dox and were cultured without hormone prior to ChIP. Cells were then crosslinked with 1% formaldehyde and chromatin sonicated to 300-500 bp. ChIP was carried out using Protein A/G Dynabeads (Thermo Fisher Scientific) coupled to antibodies against N-terminal AR (N-20; Santa-Cruz Biotechnology), C-terminal AR (Clone SP242; Spring Bioscience), ARv7 (H6 253, Epitomics), H3K27ac (C15410196, Diagenode) or FOXA1 (Mix of ab23738 and ab5089; Abcam). ChIP DNA was purified using the PCR purification kit (Qiagen). ChIP-re-ChIP was carried out as described for ChIP, but releasing the ChIP DNA with 10 nM DTT (30 min incubation at 37 C) prior to subsequent ChIP with a different antibody. ChIP-seq libraries were generated using the ThruPLEX DNA-seq Kit (Rubicon Genomics) according to the manufacturer’s instructions and standard 8 bp Illumina primers. Libraries were pooled and sequenced on the Illumina NextSeq 500 platform at the Molecular Biology Core Facility (DFCI). r> FRET and FRAP
    N-terminal EYFP-AR and C-terminal ECFP-AR were previously generated (van Royen et al., 2012). C-terminal ECFP-ARv7 was generated by cloning CFP into pCerulean-(GA)6-ARv7. N-terminal ARv7-EYFP was generated by cloning YFP into pARv7-(GA)6-Venus. The AR R585K mutation was generated by site-directed mutagenesis of pEYFP-AR-ECFP, using the following primers:
    5’-GTCTTCTTCAAAAAAGCCGCTGAAGGG-3’ (forward)
    5’-CCCTTCAGCGGCTTTTTTGAAGAAGAC-3’ (reverse) To obtain single-tagged DBD mutant AR, the DBD of pYFP-(GA)6-AR was replaced with R58K-DBD from pEYFP-R585K_AR- ECFP. The ARv7-R585K mutant was generated by site-directed mutagenesis of pYFP-(GA)6-ARv7 using primers:
    5’-AGCAAAAATGATTGCACTATTGAT-3’ (forward)
    5’-GGCGCACAGGTACTTCTGTTT-3’(reverse)
    Plasmids were transfected in the presence of 1 nM DHT into Hep3B cells using FuGENE6 (Promega). Co-expression of ARfl and ARv7 was confirmed for each cell measurement, based on CFP signal intensities. Confocal live cell imaging was performed using a Zeiss LSM510 confocal laser scanning microscope equipped with a Plan-Neofluar 40x/1.3 NA oil objective (Carl Zeiss). In acceptor photobleaching experiments (abFRET), CFP and YFP were scanned once before photobleaching (CFP ex: 458 nm, em: 470-500 nm BP; YFP ex: 514 nm, 560 nm LP). YFP was then bleached 25 times at 100% laser power at 514 nm. New images were collected after photobleaching to calculate apparent abFRET eficiency. For FRAP, YFP was scanned 1.9 msec/line of 512 pixels. A 10-pixel wide strip was selected in the middle of the nucleus for photobleaching. The fluorescent signal was monitored at 21 msec time intervals by scanning the region of interest for 40 secs at low excitation. When 200 iterations were completed, the strip was bleached for 2 iter-ations at maximum intensity.