Brane. Membranes were probed with a-AR(PG-21) followedModeling Truncated AR in AD Backgroundby HRP-conjugated a-Rabbit developed with SuperSignal and imaged on a Cell Biosciences western blot imager.Luciferase AssayLN/TC-AR cells were seeded to 24-well plates at a density of 60 K/well and incubated overnight at 37 C. The following day, cells were co-transfected with pPSA6.0-luc (180 ng/well) and pH 48-ren (20 ng/well) using Effectene Transfection Reagent (Qiagen) and treated with either DHT or various concentrations of doxycycline. Forty-eight hours post-transfection/treatment, cells were lysed and assayed for luciferase and Genz-644282 biological activity renella production using the Dual-luciferase Assay Kit (Promega) as recommended by the manufacturer on an EG G Berthold LB96V MicroLumatPlus microplate luminometer (Perkin-Elmer-Wallace, Inc.). All sample groups were completed in triplicate (mean values are reported) and have been normalized for transfection efficiency using the renella relative light units (RLU) acquired for each sample.were plated into each insert in RPMI1640 with 0.5 BSA and treated with DHT 1 nM, Dox 4.5 ng/mL, Dox 20 ng/mL and vehicle only as control. Individual treatments were completed in duplicate. 500 mls of RPMI1640 supplemented with 10 FBS was added to bottom chambers and cells were incubated for 48 hours. Cells/media from the top side of each insert were removed and inserts were incubated in 225 mls of Cell Detachment Solution for 30 minutes at 37uC. 75 mL of Lysis Buffer/CyQUANTH GR Dye Solution was added to each well containing Cell Detachment Solution and inserts were incubated in it for 15 minutes at room temperature. 200 mL of each mixture was added to one well of a 96-well plate and fluorescence absorbance was measured by a fluorescence plate reader with a 480/520 nm filter set.MicroarrayLN/TC-AR cells were treated with DHT 1 nM, Dox 4.5 ng/ mL, Dox 20 ng/mL and vehicle only as control in 10 CDT media for 24 hours after 3 days in 10 CDT media. Total RNA was extracted by using QIAshredder and RNeasy Mini Kit and following the protocols from QIAGEN. Two independent experiments were performed and RNA samples were submitted to the UC Davis ASP2215 custom synthesis Cancer Center Gene Expression Resource facility. Microarray analysis was done using Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix, South San Francisco, CA). Analysis of the expression data from the Dox 4.5 ng/mLtreated cells, Dox 20 ng/mL-treated cells and DHT-treated cells versus the vehicle-treated cells was conducted using DNA-Chip Analyzer software. Filter criteria are p#0.05, signal log ratio 0.6 1407003 (1.5fold) and present (P) for detection. Gene Ontology (GO) analysis was performed by DAVID Functional Annotation tools (http://david.abcc.ncifcrf.gov/home.jsp) and Gene Set Analysis Toolkit [16].ImmunostainingLN/TC-AR cells were plated to 6-well plates with one slide in each well and were hormone-depleted for 3 days then treated with 4.5 ng/mL doxycycline treatment or vehicle only as control for 24 hours. Slides were washed with 16 PBS twice and fixed in 4 paraformaldehyde at 4uC for 30 minutes. After three washes with 16 PBS, slides were permeabilized with 1 Triton X-100 in 16 PBS followed by 1 NP-40 in 16PBS for 10 minutes. Slides were blocked with 2 BSA in TBST for 30 minutes then washed 3 times with 16 PBS. Anti-FLAG M2 antibody was diluted 1:50 in 2 BSA in TBST and incubated with slides for one hour at room temperature (RT). Slides were then washed with 16 PBS 3 times and incuba.Brane. Membranes were probed with a-AR(PG-21) followedModeling Truncated AR in AD Backgroundby HRP-conjugated a-Rabbit developed with SuperSignal and imaged on a Cell Biosciences western blot imager.Luciferase AssayLN/TC-AR cells were seeded to 24-well plates at a density of 60 K/well and incubated overnight at 37 C. The following day, cells were co-transfected with pPSA6.0-luc (180 ng/well) and pH 48-ren (20 ng/well) using Effectene Transfection Reagent (Qiagen) and treated with either DHT or various concentrations of doxycycline. Forty-eight hours post-transfection/treatment, cells were lysed and assayed for luciferase and renella production using the Dual-luciferase Assay Kit (Promega) as recommended by the manufacturer on an EG G Berthold LB96V MicroLumatPlus microplate luminometer (Perkin-Elmer-Wallace, Inc.). All sample groups were completed in triplicate (mean values are reported) and have been normalized for transfection efficiency using the renella relative light units (RLU) acquired for each sample.were plated into each insert in RPMI1640 with 0.5 BSA and treated with DHT 1 nM, Dox 4.5 ng/mL, Dox 20 ng/mL and vehicle only as control. Individual treatments were completed in duplicate. 500 mls of RPMI1640 supplemented with 10 FBS was added to bottom chambers and cells were incubated for 48 hours. Cells/media from the top side of each insert were removed and inserts were incubated in 225 mls of Cell Detachment Solution for 30 minutes at 37uC. 75 mL of Lysis Buffer/CyQUANTH GR Dye Solution was added to each well containing Cell Detachment Solution and inserts were incubated in it for 15 minutes at room temperature. 200 mL of each mixture was added to one well of a 96-well plate and fluorescence absorbance was measured by a fluorescence plate reader with a 480/520 nm filter set.MicroarrayLN/TC-AR cells were treated with DHT 1 nM, Dox 4.5 ng/ mL, Dox 20 ng/mL and vehicle only as control in 10 CDT media for 24 hours after 3 days in 10 CDT media. Total RNA was extracted by using QIAshredder and RNeasy Mini Kit and following the protocols from QIAGEN. Two independent experiments were performed and RNA samples were submitted to the UC Davis Cancer Center Gene Expression Resource facility. Microarray analysis was done using Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix, South San Francisco, CA). Analysis of the expression data from the Dox 4.5 ng/mLtreated cells, Dox 20 ng/mL-treated cells and DHT-treated cells versus the vehicle-treated cells was conducted using DNA-Chip Analyzer software. Filter criteria are p#0.05, signal log ratio 0.6 1407003 (1.5fold) and present (P) for detection. Gene Ontology (GO) analysis was performed by DAVID Functional Annotation tools (http://david.abcc.ncifcrf.gov/home.jsp) and Gene Set Analysis Toolkit [16].ImmunostainingLN/TC-AR cells were plated to 6-well plates with one slide in each well and were hormone-depleted for 3 days then treated with 4.5 ng/mL doxycycline treatment or vehicle only as control for 24 hours. Slides were washed with 16 PBS twice and fixed in 4 paraformaldehyde at 4uC for 30 minutes. After three washes with 16 PBS, slides were permeabilized with 1 Triton X-100 in 16 PBS followed by 1 NP-40 in 16PBS for 10 minutes. Slides were blocked with 2 BSA in TBST for 30 minutes then washed 3 times with 16 PBS. Anti-FLAG M2 antibody was diluted 1:50 in 2 BSA in TBST and incubated with slides for one hour at room temperature (RT). Slides were then washed with 16 PBS 3 times and incuba.
