Supplemental material #1
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Supplemental material #2
Antibodies used for Western blot analysis and technical information.
Whole cell protein extracts were prepared using RIPA buffer as described elsewhere (Rosenberg, 1996), resolved by SDS-PAGE on 10% gels and transferred to nitrocellulose membranes (BioRad, Hercules, CA). The following antibodies were used: anti-phospho-Mek1/2, anti-phospho-Erk1/2, anti-phospho-SAPK/JNK, anti-phospho-c-p38, anti-SAPK/JNK, anti-Erk1/2, anti-Mek1/2, and anti-phospho-c-Myc (all from Cell Signaling Technology, Beverly, MA); anti-p38, anti-p21, anti-p27, anti-Cyclin D1, anti-GR and anti-actin (all from Santa Cruz Biotechnology, Pasadena, CA); anti-Ki67 antibodies (Abcam, Cambridge,MA ), anti-PARP, anti-Maspin and anti-AMACR (BD Pharmingen,San Jose, CA); anti-hepsin (Cayman, Ann Arbor, Michigan) and anti-V5 tag (Invitrogen Corpor., Carlsbad, CA). Membranes were blocked with 5% Blotto in TBS, incubated with primary antibodies overnight at 4C, followed by peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology, Beverly, MA). ECL reagent (Amersham Pharmacia Biotech, Sweden) was used for the band visualization. To verify equal loading and adequate transfer, the membranes were probed with anti-actin and/or anti--tubulin antibodies (Santa Cruz Biotechnology, Pasadena, CA).To quantify the signals, images were scanned and digitized using ImageJ software (NIH, Bethesda, MD).
Supplemental material #3
Source of reporter plasmids used in transient transfections and Luciferase assay.
The following Fireflight Luciferase reporters were used: TAT3-Luc (provided by Dr. K.R.Yamamoto, University of California, San Francisco, CA), x5 B-Luc (provided by Dr. W.C. Greene, Gladstone Institute for Virology and Immunology, University of California, San Francisco, CA); ISRE-Luc and STAT3-Luc (provided by Dr. D.J. Klumpp, Northwestern University, Chicago IL). Reporters for STAT-1, NFAT-c, SRF, Ets-1, and Elk-1 were obtained from Panomics (Panomics Inc., Fremont, CA).
Supplemental material #4
Prostate marker primer sequences for RT-PCR.
The following primer pairs were used:
1.Maspin: forward 5’-GTCACAGTGGACTAATCCCAGC-3’; reverse 5’-GCACCTCTATGGAATCCCCA-3’;
2.Hepsin: forward 5’-CGCTCGGCTCATGGTCTTT-3’; reverse 5’-GCAATCACACACGGAGATGAC-3’;
3.AMACR: forward 5’-AAATGGTTATCATTAGGGCTTTTGA-3’: reverse 5’-TTCCTTTTTCACTAGAACCCATTCA-3’;
4.GAPDH: forward 5’-GTGAAGGTCGGAGTCAACG-3’; reverse 5’-TGAGGTCAATGAAGGGGTC-3’.
Supplemental material #5
Immunostaining of prostate tissues and quantitative analysis of staining.
Tissues were obtained from two cohorts of consented untreated patients (ages 40-82) by TURP (transurethral prostatic resection) or radical prostatectomy. All patients signed consent forms approved by IRB at NorthwesternUniversity or N.N.BlokhinCancerResearchCenter of RussianAcademy of Medical Sciences (Moscow, Russia). One hundred two prostate specimens were harvested at the RussianCancerCenter. The diagnoses were verified and GR staining analyzed by Dr. A. Karseladze. One hundred sixteen prostate specimens were from the tissue core facility at the R.H.LurieComprehensiveCancerCenter, NorthwesternUniversity (Chicago, IL). The diagnoses were verified and GR staining analyzed by Dr. X. Yang. Immunostaining of paraffin-embedded sections of formalin-fixed prostate samples was performed with Envision+ System-HRP (DAB) kit according to the manufacturer’s protocol (DakoCytomation, Carpinteria, CA). After Ag retrieval (5 minutes at 20-25 psi in citric buffer, pH 6.0), the sections were treated 20 minutes with 3% H2O2, blocked with 20% goat serum in PBS, and incubated with primary mouse polyclonal Ab against GR (Novocastra, Norwell, MA) followed by secondary anti-mouse IgG-reagent provided in the Envision+ System-HRP (DAB) kit. Immunostaining was visualized with DAB chromogen (DakoCytomation, Carpinteria, CA) and sections were counterstained in Mayer's hematoxylin. Human skin and blood vessel samples were used as a positive control. In both cohorts the number of prostate epithelial cells with nuclear GR signal was evaluated by + to +++ scoring: – equals absent, + equals weak (1-25% positive cells), ++ equals moderate (26-50% positive cells), and +++ equals strong (51-100% positive cells) staining. The percentage of specimens with the same diagnosis stained at a given intensity was determined.
Technical information for immunostaining of cell cultures.
Cells plated on sterile coverslips in 24-well plates (2.0x104 cells/well) were treated for 1-3 days with 10-9-10-6 M FA. The cells were fixed 15 minutes in 2% formaldehyde, permebealized for 3 hours at -20C with 50%:50% acetone: methanol mixture, washed in PBS and blocked for 1 hour in 20% donkey serum. Primary antibodies were applied overnight at 4C. Anti-rabbit donkey FITC-conjugated and/or anti-mouse donkey Cy-3 conjugated secondary antibodies (both from Jackson Immunoresearch, West Grove, PA) were applied and the staining analyzed using Zeiss Axioplan2 microscope. DAPI (Vector Laboratories Inc, Burlingame, CA) was used to identify the nuclei.
Supplemental material #6
Technical information for transcription factor Protein/DNA Arrays.
To simultaneously evaluate the activity of multiple TFs we used Combo-Array version of TranSignalTM Protein/DNA interaction array (Panomics Inc., Fremont, CA) containing probes for binding sites for over 300 TFs (for detailed description see Jiang et al., 2004). LNCaP-GR and LNCaP-V cells plated onto 10 cm dishes in complete media were treated at 50% confluence with 10-7 M FA or vehicle for 3 days. Nuclear protein extracts were prepared using Nuclear Protein Isolation Kit (Panomics. Fremont, CA) according to the manufacturer’s protocol, and protein concentrations were measured with BSA Protein Reagent (BioRad, Hercules, CA). Four µg of nuclear protein were incubated with biotinylated oligonucleotide probes (15 minutes at 30C). Protein-bound probes were isolated and hybridized on the Combo-Array membranes (42C overnight). The membranes were sequentially washed in 2% SSC/0.5% SDS and 0.1% SSC/0.5% SDS (42C, 20 minutes). Membrane-bound biotinylated probes were detected by incubation with Streptavidin–HRP and luminescent substrate (Panomics Inc, FremontCA). The experiment was repeated three times. The scanned images representing DNA-binding signals were analyzed with ImageJ software (National Institute of Health, BethesdaMD). The differences in signal between FA- and vehicle-treated samples ≥2 were considered statistically and biologically significant if they were revealed in all three experiments.