Cell culture and hormone treatment
LNCaP (ATCC CRL-1740) and CWR22Rv1 (ATCC CRL-2505) prostate cancer cells were cultured in RPMI media. All cells were grown in media supplemented with 10% FCS and 100ug/ml penicillin/streptomycin in a 37°C incubator with 5% CO2. For hormone treatment, cells were grown to 60-80% confluency and then serum starved overnight in either serum-free media or media supplemented with 5% charcoal-dextran stripped FBS RPMI. The synthetic androgen methyltrienolone (R1881) (Perkin Elmer, Boston, MA) was then added to the charcoal-dextran stripped FBS RPMI media and cells were treated with 5nM R1881 for 24 hours unless otherwise noted in figure legends. For inhibition of AR, 10μM bicalutamide/casodex (LKT Labs, St. Paul, MN) was added 2 hours prior to treatment with R1881.
Two million cells were treated with formaldehyde to crosslink proteins to DNA and lysed as per manufacturer’s recommendations using Millipore EZ ChIP Assay (Upstate Biotechnology Inc., Billerica, MA). Chromatin was sheared by sonication (Biosonik III, Bronwill Scientific, Rochester, NY) to fragments of 200–1,000 bp in length. The supernatant was pre-cleared by incubation with Protein G Agarose and incubated overnight at 4°C with either anti- AR (Santa Cruz), Sp1 (Santa Cruz) and control polymerase II antibodies or non-immune IgG (Upstate Biotechnology Inc.). The complexes were recovered from Protein G magnetic beads, crosslinks were reversed and DNA was purified. Four percent of both immunoprecipitated and input chromatin were amplified by PCR using Taq polymerase (Applied Biosystems by Roche Molecular System, Inc) and the appropriate primers (ARE I (FOR): 5′-TTCGAGAGTGAGGACGTGTG-3′, ARE I (REV): 5′-AGGGAGCA GGAAA GTGAGGT-3′, ARE II (FOR): 5′-TCACTGACTAACCCCGGAAC-3′, ARE II (REV): 5′-TTTGG GACTGGAGTTGCTTC-3′, ARE III (FOR): 5′-GGCTCTTTTAGGGGCTGAAG-3′, ARE III (REV): 5′-AGGCTGATGAACGGGATATG-3′, VEGF V88 (FOR) 5′-CCGCGGGCGCGTGTC TCTGG-3′, VEGF V88 (REV) 5′-TGCCCCAAGCCTCCGCGATCCTC-3′). Following an initial 10 min denaturation at 95°C, DNA was amplified by 32–35 cycles of: 1) 20 sec denaturation at 95°C, 2) 30 sec annealing at either 52°C (for ARE III primers), 53°C (for ARE II primers), 58°C (for ARE I and VEGF V88 primers) and 3) 30 sec extension at 72°C; amplification was completed with a 2 min final extension at 72°C. PCR products were electrophoresed on 1% agarose gel, and ethidium bromide stained DNA was visualized by a gel doc system (Biorad, Hercules, CA).
For quantitation of immunoprecipitated chromatin by quantitative real-time PCR (qRT-PCR), purified DNA samples were amplified in an ABI 7000 thermocycler using primers listed above and following manufacturer’s recommendation for SYBR Green Q-PCR (Applied Biosystems, Foster City, CA). Each PCR reaction was carried out in triplicate and average Ct values were normalized to total input (non- immunoprecipitated) DNA. The amount of DNA immunoprecipitated with the target antibody from hormone treated cells R1881 was compared to that of control samples treated only with vehicle. Shown are input normalized Ct values from chromatin of treated cells relative to untreated control cells.
RNA isolation and quantitative real-time PCR
RNA was isolated from subconfluent cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma, St. Louis, MO) as per manufacturer’s recommendation. Following quantitation, 1μg of RNA was reverse transcribed using the High Capacity cDNA ReverseTranscription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using either Taqman Universal Master Mix with pre-designed Taqman Gene Expression Assay probe sets for VEGFA (Hs00900057_m1) and 18S (Hs99999901_s1) or SYBR Green Master Mix with primers specific for VEGF, Sp1, GAPDH, and Beta-actin: VEGF (FOR): 5′-CGAAACCATGAACTTTCTGC-3′, VEGF (REV): 5′-CCTCAGTGGGCACACACTCC-3′, Sp1 ( FOR) 5′-TGCATTTCAAGGAATGGAAT-3′, Sp1 (REV) 5′-GCTTCCTTGGTGTGAAGAGA-3′, GAPDH (FOR): 5′-CCATCACCATCTTCCAGGAG-3′, GAPDH (REV): 5′-GGATGATGTTCTGGAGAGCC-3′, Beta-actin (FOR): 5′-GTGGGGCGCCCCA GGCACCA-3′, Beta-actin (REV): 5′-GTCCTTAATGTCACGCACGATTTC-3′). The comparative Ct method  was used to analyze gene expression differences between control (untreated) cells and cells treated with R1881 alone or with the anti-androgen casodex.
Subconfluent monolayers of LNCaP and 22Rv1 cells were washed in PBS and proteins were extracted using RIPA lysis buffer (1% NP40, 0.5% Na Deoxycholate, 0.1% SDS, and 150 mM NaCl) containing protease inhibitors. To quantify the amount of proteins present in each lysate, bicinchoninic acid (BCA) assays (Pierce, Thermo Fisher Scientific, Rockford, IL) were performed and absorbance was measured at 600nm on a Dynex Technologies (Chantilly, VA) MRX Revelation plate reader. Proteins (25-50ug) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a Polyvinylidene Fluoride (PVDF) membrane and blocking with 5% casein, blots were probed overnight at 4°C with polyclonal VEGF and AR antibodies (Santa Cruz), and monoclonal β-actin antibody (GenScript). Washed blots were then incubated for 1 hr in either HRP-conjugated anti – rabbit (GenScript) or anti – mouse (Santa Cruz) antibodies. Proteins were visualized by incubating the membrane in a luminol ECL solution followed by chemiluminescent detection using a Fuji LAS 3000 (GE, Piscataway, NJ) detection system. Bands were quantified using ImageJ analysis and normalized to actin levels.
Nuclear extracts from 22Rv1 cells were prepared using Active Motif’s Universal Magnetic Co-IP kit (Carlsbad, CA) as per manufacturer’s recommendations. Cells were swelled in Hypotonic Buffer containing phosphatase-, deacetylase-, and protease- inhibitors, then lysed in 5% detergent. This suspension was then centrifuged at 14,000 x g and the supernatant was discarded leaving the nuclear fraction which was enzymatically sheared in the presence of the same inhibitors. Nuclear extracts (150-200μg) were then combined with 5μg of either AR (Santa Cruz) or Sp1 (Upstate) antibodies, or negative control IgG in the presence of the same inhibitors. Following antibody incubation, complexes were pulled down with Protein G magnetic beads. After washing, these complexes were separated by SDS-PAGE and identified by Western blot analysis (as described above).
Plasmid Transfection and Luciferase Assay
The pGL3-VEGF luciferase reporter constructs (V88, V411, or V2274) were generously provided by Dr. Xie  and DNA purified by the Qiagen plasmid Maxi prep kit (Qiagen, Valencia, CA). LNCaP and 22Rv1 cells were plated in 12-well plates as described above. After reaching ~70-90% confluency, cells were serum-starved for 18–24 hours with serum-free RPMI and then transfected with VEGF reporter constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described . After 4–6 hours, transfection media was replaced with appropriate growth media. For hormone induction, 5nM R1881 was added to media with 5-10% charcoal-dextran stripped FBS as described above. After 48 hours cells were lysed and luciferase activity was measured using a Promega luciferase assay kit (Promega, Sunnyvale, CA) and a Turner luminometer (Promega, Sunnyvale, CA) following manufacturer’s recommendations. The luciferase activity was normalized to total cell protein, using a micro BCA protein assay, as described above. All experiments were done in triplicate and repeated at least three times. Standard errors of the mean were determined using GraphPad InStat software (San Diego, CA). Significance was determined by Student’s t-test.
Site directed mutagenesis
Potential binding sites in the VEGF promoter were identified using MatInspector, as previously described . Predicted AR and Sp1 binding sites in the VEGF promoter construct were then mutated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Agilent Technologies, Santa Clara, CA). Primers were designed according to the manufacturer’s suggestions using the QuikChange Primer Design Program. Primers containing the desired mutation (shown in bold) are listed below:
(ARE I (FOR):
5′-CTCTATCGATAGGTACCGTGGTCAG CTCTCCCC ACCCGTC CCTGTC-3′,
ARE I (REV): 5′GACAGGGACGGGTGGGGAGAGCTGAC CACGGTACCTATCGA TAGAG-3′,
ARE II (FOR): 5′-GGAACCACACAGCTTCCCACTG TCAGCTCCACA AAC TTGG-3′,
ARE II (REV): 5′-CCAAGTTTGTGGAGCTGACAGT GGGAAGCTGTGTGGTTCC-3′,
ARE III (FOR): 5′-GCCCCAAGATGTCTACAGCTTACGG TCCTGGGGTGC-3′,
ARE III (REV): 5′-GCA CCCCAGGACCGT AAGCTGTAGACATCTTGGGGC-3′,
Sp1.2/Sp1.3 (FOR): 5′-GCCCC CCG GTT CGGGCCGGGTT CGGGGTCCC-3′,
Sp1.2/Sp1.3 (REV): 5′-GGGACCCCG AA CC CGG CCC GAA CCGGGGGGC-3′,
Sp1.4 (FOR): 5′-GGGTCCCGGCGGTT CGGAGCCATGCG-3′,
Sp1.4 (REV): 5′-CGCATGGCTCCGAA CCGCCGGGACCC-3′).
PCR was performed using the V88, the V411, or the V2274 luciferase reporter constructs and the appropriate mutant primers. After PCR amplification, parental strands were digested with DpnI and XL1-Blue super competent cells were transformed with remaining mutant DNA. Individual colonies were grown, plasmids were purified (Qiagen, Valencia, CA) and sequenced to verify that the correct base pairs were changed (Cleveland Clinic Genomics Core, Lerner Research Institute, Cleveland, OH). Luciferase assays were performed using mutant constructs as described above.