ed with RNAi oligonucleotides by using DharmaFECT 3 AEB-071 web transfection reagent for a final concentration of 50 10760364 nM. 22RV1 cells were transfected with siRNA oligonucleotides by using Lipofectamine2000 transfection reagent for a final concentration of 50 nM. Cells were harvested at indicated time points post-transfection. R1881 was resuspended in 100% ethanol. MDV3100 was purchased and resuspended in DMSO. JQ1 was purchased from Sigma and resuspended in DMSO. Cell growth was determined at the end of treatment with the trypan blue exclusion method following manufacturers’ instructions. All cell culture experiments were performed with biological triplicate samples and confirmed in repeat experiments. LNCaP cells with stable overexpression of c-Myc or empty vector were generated by transfecting LNCaP cells with pCDNADEST40-c-Myc or pCMV6-AN-His and selecting with G418. 6 AR and c-Myc Promote Prostate Cancer Progression Colony-formation Assay 200,000 LNCaP cells with stable overexpression of empty vector or c-Myc were plated in 10 cm dishes. RPMI with 10% charcoal-stripped fetal bovine serum supplemented with 300 ug/ ml G418 and 10 ug/ml bicalutamide treatment was added to the cells every other day for 14 days. Next, the cells were fixed with 4% formaldehyde and stained with syto60. Images were taken with Licor Odyssey imaging system. Cruz); actin and c-Myc. Secondary antibodies were purchased from Licor. QRT-PCR RNA was extracted from cell pellets stored in RNAlater reagent using a MagMAX Total RNA Isolation kit or Trizol according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer. 250 ng1 ug RNA was reversetranscribed using a High-Capacity cDNA Reverse Transcription kit with random primers. Realtime PCR was performed using a 7500 Fast thermocycler with the following cycling program: 50uC for Immunoblotting Immunoblotting experiments were performed as described 10760364 previously. Final images were obtained with Licor Odyssey imaging system. Primary antibodies used were: AR Immunoblotting was performed to determine the protein levels of c-Myc. B) QRT-PCR was performed to determine the mRNA level of c-Myc and c-Myc targets genes KIF11, CDKN1A, TPX2, and AURKB relative to actin. denotes p,0.05 compared to vehicle. C) Cell viability was determined at the end of treatment with the trypan blue exclusion method. p,0.01 for the 250 nM and 500 nM doses vs. vehicle in all three cell lines. doi:10.1371/journal.pone.0063563.g006 8 AR and c-Myc Promote Prostate Cancer Progression 2 min, 95uC for 10 min, 40 cycles of 95uC for 15 sec dissociation, 60uC for 1 min annealing/extension/read. 10 mL singleplex RTPCR reactions contained 1X Taqman universal standard mastermix, 1X Taqman hydrolysis probe, and 10 ng RNAequivalent cDNA template. Human beta-actin was used as an endogenous control. Primer information is included in Chromatin Immunoprecipitation 5 mg of anti-AR antibody, anti-acetylated histone H3 antibody or normal rabbit antibody were added to sheared, formaldehyde crosslinked chromatin derived from 1 to 26106 cells to immunoprecipitate DNA overnight at 4uC. 1% of chromatin was removed prior to immunoprecipitation as input. Immune complexes were collected with protein A magnetic beads. After extensive washing, immune complexes were released, cross-links were reversed, and DNA was purified with mini-elute PCR purification kit and eluted with 60 ml EB. Realtime QPCR was performed as described above using 1