PMC

COS-7 cells were transfected with the AR expression vector and the constitutively active Ack1 expression vector and incubated for 48 hrs. Cells were fixed and AR expression detected with the AR monoclonal antibody and FITC-conjugated secondary antibody. Nuclear staining was performed with DAPI. Original magnifications, 1,000X.

(A, B) LNCaP cells stably overexpressing full length AR were treated with EGF (100 ng/ml) or heregulin (10 ng/ml) or Gas6 (100 ng/ml) for 1 hr, as indicated. Cell lysates were subjected to immunoprecipitation with the FLAG antibody, followed by immunoblotting with the phospho-specific AR or total AR antibody. (C, D) LNCaP cells stably overexpressing truncated AR were treated with EGF (100 ng/ml) or heregulin (10 ng/ml) or Gas6 (100 ng/ml) for 1 hr, as indicated. Cell lysates were subjected to immunoprecipitation with the FLAG antibody, followed by immunoblotting with the phospho-specific AR or total AR antibody.

(A) COS-7 cells were transfected with the AR expression vector and the constitutively active Ack1 L487F expression vector [] and incubated for 48 hrs. The cells were serum-starved overnight and treated with or without DHT (10 nM) for 2 hrs. Subcellular fractionation was performed. Fifteen μgrams of protein from cytoplasmic (C) and nuclear (N) fractions were immunoblotted with the AR antibody. Laminin A/C and 14-3-3 β were used as markers of nuclear and cytoplasmic fractions, respectively. (B) COS-7 cells were transfected with the AR expression vector (full length or truncated) and incubated for 48 hrs. Subcellular fractionation and immunoblotting were performed as described above.

(A) LNCaP cells stably overexpressing vector or AR constructs were grown in phenol red free media and serum starved for overnight and treated with or without 10 nM DHT for 2 hrs. Cell fractionation was performed. The cytoplasmic and nuclear lysates from LNCaP cells expressing full length AR constructs were subjected to immunoprecipitation with FLAG antibody, followed by immunoblotting with AR antibody, as indicated. Laminin A/C and 14-3-3 β were used as markers of nuclear and cytoplasmic fractions, respectively. (B) The cytoplasmic and nuclear lysates from LNCaP cells stably expressing truncated AR constructs under androgen-deprived conditions were subjected to immunoprecipitation with FLAG antibody, followed by immunoblotting with AR antibody, as indicated. Laminin A/C and 14-3-3 β were used as markers of nuclear and cytoplasmic fractions, respectively.

LNCaP cells stably overexpressing AR constructs or vector control were derived by retrovirus-mediated transduction and antibiotic selection. (A) Lysates from LNCaP cells overexpressing full-length AR were subjected to immunoprecipitation with the AR antibody, followed by immunoblotting with the AR antibody or the epitope tag HA antibody, as indicated. Total cell lysates were also immunoblotted with the AR monoclonal antibody and actin antibody. (B) The mRNA levels of AR was determined by quantitative reverse transcription PCR of total RNA isolated from LNCaP cells stably overexpressing full-length AR. (C) Lysates from LNCaP cells stably overexpressing truncated AR were subjected to immunoprecipitation, followed by immunoblotting, as indicated. Total cell lysates were also immunoblotted with the AR monoclonal antibody and the actin antibody. (D) The mRNA levels of AR was determined by quantitative reverse transcription PCR of total RNA isolated from LNCaP cells stably overexpressing truncated AR.

(A, B) LNCaP cells were transiently transfected with vector alone or the expression vector encoding full-length AR wt, truncated AR wt (TR-AR-WT), Y267F or Y363F mutants of truncated AR along with the AR-dependent reporter MMTV-luciferase (A) or ARR2-PB-luciferase (B). After 48 hours in androgen depleted media, luciferase activity was determined from cell lysates. Data shown with the mean + standard deviation of triplicate samples are representative of three independent experiments with similar results. *, p<0.001 vs TR-AR-WT; **, p<0.05 vs TR-AR-WT by two-group t-test adjusted using the Bonferroni method. (C) Cell lysates of COS-7 cells transiently transfected with respective AR constructs were immunoblotted with the monoclonal antibody against AR.

(A, B) LNCaP cells stably overexpressing full length AR (A) or truncated AR (B) were grown in a 96-well plate for 72 hours in media with charcoal-stripped serum and 0, 0.1, 1, or 10 nM of DHT, as indicated. Absorbance at 450 nm was measured after incubation with the colorimetric dye WST-8 for 4 hours. Values were normalized to vector control cells treated with 0 nM DHT. Data shown are the mean + standard deviation of three independent experiments, each performed with triplicate or quadruplicate wells. *, p<0.001 vs AR-WT; **, p<0.05 vs AR-WT by two-group t-test adjusted using the Bonferroni method. (C, D) Cells stably overexpressing full length AR (C) or truncated AR (D) were plated in soft agar and incubated for 3 weeks. Colonies were stained and counted. A representative view of the colonies is shown below. Data shown are the mean + standard deviation of two (C) or three (D) independent experiments with triplicate samples. *, p<0.001 vs AR-WT; **, p<0.05 vs AR-WT by two-group t-test adjusted using the Bonferroni method.

(A, B) LNCaP cells expressing truncated AR constructs were grown in phenol red free media supplemented with charcoal-stripped serum. Chromatin immunoprecipitation analysis for binding of FLAG-tagged truncated AR protein to the androgen response element III enhancer region of PSA (A) and KLK2 (B) genes were performed. The amount of precipitated DNA was determined by quantitative PCR. The data shown are the mean and standard deviation of three independent experiments performed in triplicate. *, p<0.001 vs TR-AR-WT by two-group t-test adjusted using the Bonferroni method. (C, D) mRNA levels of PSA and KLK2 from LNCaP cells expressing truncated AR constructs under androgen-deprived conditions as above were determined by quantitative RT-PCR performed in triplicate. The data shown are representative of two independent experiments. *, p<0.001 vs TR-AR-WT; **, p<0.05 vs TR-AR-WT by two-group t-test adjusted using the Bonferroni method. (E) Microarray gene expression analysis from LNCaP cells expressing truncated AR constructs under androgen-deprived conditions using vector control as reference was performed. The AR transcriptional pathway activity score was calculated using an established androgen response signature []. The score from two independent samples is shown. A positive score indicates increased activation of the AR transcription pathway while a negative score indicates decreased activation of the pathway.