triplicate on cDNA and gDNA templates using Platinum SYBR Green qPCR Super Mix-UDG with ROX (Invitrogen, 11744-500) as previously described [44]. PCR using 1 ml of 20-fold diluted cDNA was performed for 30 cycles with Phusion High Fidelity DNA Polymerase (Finnzymes, F-530S). All primer sequences are listed in Table S3. RT-PCR products were initially analyzed on 2– 3% agarose gels, and subsequently using the QIAxcel capillary gel electrophoresis system (Qiagen), which was also used for image acquisition. Ratios between products representing transcripts of interest multiplexed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) controls as well as mRNA isoforms were determined using QIAxcel software. The relative normalised gene expression was calculated as a ratio of the fluorescent intensity of the band representing amplification of the transcript of interest/fluorescent intensity of band representing amplification of GAPDH). The relative normalised expression ratio was calculated as follows: fluorescent intensity of band representing the alternative (andro- gen-regulated) transcript/fluorescent intensity of band or fluores- cent intensity of band representing 39 transcript end/59 transcript end. The independent sample T-Test was employed to identify statistically significant differences in total transcript and alternative mRNA isoform expression with p,0.05 taken to indicate statistical significance. To confirm the identities of RT-PCR products distinct gel bands were purified with the QIAquick Gel Extraction kit (Qiagen, 28704), cloned using a CloneJET PCR Cloning Kit (Fermentas Life Sciences, K1231) and positive transformants were sequenced (GATC Biotech) using a T7 sequencing primer. Chromatin Immunoprecipitation (ChIP) assays ChIP assays were performed as previously described [44], using 2 mg anti-AR antisera and normal IgG controls. qPCR (Applied Biosystems 7900HT) was performed on inputs and recovered material to assess AR association to DP (downstream promoter) 1 and 2, and non-AR-binding region within GREB1 (chromosome positions chr16:2132598–2132747, chr16:2133122–2133271, and chr2:11597650–11597669, respectively). All experimental results shown are the means of at least three independent experiments 6 standard error (SE). Supporting Information Materials and Methods S1 (DOCX) Figure S1 Analysis of androgen-regulated gene expres- sion. (A) Experimental validation of changes in KLK3 gene expression. LNCaP cells were grown in steroid-deplete medium for 72 hours followed by treatment (n = 4) or absent (n = 4) for 24 hours with 10 nM R1881. KLK3 and GAPDH transcript expression was analysed by qPCR analysis KLK3 transcript levels were normalised to GAPDH levels. Data from qPCR triplicates were used to obtain the mean 6 SE for each sample. (B) Principal Component Analysis (PCA) mapping of microarray datasets show segregation into control (Ctr) and androgen treatment (And) conditions. (C) Scatterplot showing gene expression in steroid- deplete medium and following androgen treatment. Differentially- expressed genes are shown in red ($2-fold change in gene expression with a q-value#0.005). (D) Gene Ontology (GO) analysis of genes up-regulated in response to androgen treatment. (EPS) Figure S2 Dose-independent androgen-regulation of alternative mRNA isoforms occurs with synthetic and natural androgens. Fluorescent quantification of capillary agarose gel electrophoretic analysis of RT-PCR products ampli- Androgen-Dependent Exons fied from mRNA from LNCaP cells grown in steroid-deplete medium or after 24 hours of treatment with different concentra- tions of synthetic (R1881) and natural (dihydrotestosterone; DHT) androgens. The relative normalised expression ratio is the contribution of the PCR product representing the alternative (androgen-regulated) transcript/constitutive transcript or 39 tran- script end/59 transcript end. Data from three independent replicates were used to obtain the means 6 SE. (TIF) Figure S3 Androgen-regulated mRNAs isoforms are expressed in benign and malignant prostate cell lines and cases of clinical PCa. (A) Capillary agarose gel electrophoretic analysis of RT-PCR products amplified from mRNA from prostate epithelial cell lines. LNCaP-AI and LNCaP- cdxR cells are able to grow in steroid-deplete medium and medium containing anti-androgens (bicalutamide), respectively. The predicted mRNA isoforms detected are shown to the right of the gel exactly as summarised in Figure 2B. (B) Fluorescent quantification of capillary agarose gel electrophoretic analysis of RT-PCR products amplified from mRNA from PCa cell lines that do not exhibit an androgen-dependent transcriptional response (PC-3, PC-3M, DU145). Cells were grown either in steroid-deplete medium or after 24 hours of treatment with 10 nM R1881. Data from three independent replicates were used to obtain the means 6 SE. (C) Agarose gel electrophoretic analysis of RT-PCR products amplified from mRNA from clinical prostate samples from patients with hormone-na¨ıve (HN) (odd lanes) and following development of castration-resistant (CR) (even lanes) disease (n = 10). The predicted mRNA isoforms detected are shown to the right of the gel exactly as summarised in Figure 2B. As a control to demonstrate mRNA integrity and overall levels of gene expression between patients, equal levels of GAPDH could be detected in each of the patient mRNA samples. Densitometric band quantification was performed to obtain the relative normalised expression ratio, which is the contribution of the PCR product representing the alternative (androgen-regulated) transcript/constitutive transcript (data not shown). M = 1 Kb plus DNA ladder (Invitrogen). (TIF) Figure S4 Expression of novel mRNA isoforms is both directly and indirectly regulated by androgens. (A) UCSC Genome Browser showing chromosomal locations and annotated transcript details for each novel androgen-regulated alternative mRNA isoform. Custom tracks (black bars below ‘‘Human_LN- CaP_AR_R1881(16h)_Chinnaiyan’’) for each gene show locations of AR binding site(s) within the LNCaP genome as determined by ChIP-Seq [34]. (TIF) Table S1 Androgen-regulated gene expression changes. Individual gene expression changes following androgen treatment are shown with fold change predicted based upon microarray prediction, and the actual fold change where measured by RT- PCR. Androgen-regulated genes identified previously published studies are indicated. The functions and associated processes of each gene are annotated where known. (XLS) Table S2 Overlap of identified androgen-regulated genes with previously-published datasets. (DOC) Table S3 Genomic sequence of oligonucleotide prim- ers. (A) qPCR for gene expression analysis (B) RT-PCR for splicing analysis (C) RT-PCR for gene expression analysis (D) PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e29088