The human tissues analyzed in this study were from patients treated at Brigham and Women’s Hospital, Dana-Farber Cancer Institute or Vall d’Hebron University Hospital in Barcelona, Spain, all of whom provided informed consent. The study was approved by the Institutional Review Board at Dana-Farber Cancer Institute. Expression analysis cDNA was prepared for expression analysis using Invitrogen SuperScript III Reverse Transcription kit. DFCI prostate samples, colon samples and breast samples were analyzed via competitive RT-PCR using Sequenom iPLEX matrix-assisted laser desorp- tion/ionization (MALDI)-time of flight mass spectrometry tech- nology. Expression levels of two MSMB isoforms and five NCOA4 isoforms were measured. These splice variants represent all isoforms reported in Ensembl genebuild 52. RNA expression of TIMM23 and three housekeeping genes (ACTB, MYL6 and RPL13A) were also measured. Primer, probe and competitor oligo sequences are available upon request. Reactions were performed in quadruplicate using 8 serial dilutions of competitor, and the EC50 was calculated using QGE Analyzer software (Sequenom). The PHS subgroup was analyzed using Illumina cDNA-mediated Annealing, Selection, Extension and Ligation (DASL) expression assay (Illumina). Expression data analysis A gene expression normalization factor was calculated using the geometric mean of expression level of the three housekeeping genes. Linear regression was used to assess whether expression levels increased (or decreased) as the number of T-alleles of rs10993994 increased; for prostate tissue, differences between prostate tumor and normal tissue levels were also assessed and random effects linear regression was used to account for within- sample correlation of tumor/normal pairs. Genotyping Genotyping of DNA from each subject was carried out using Sequenom iPLEX matrix-assisted laser desorption/ionization (MALDI)-time of flight mass spectrometry technology. Cell culture LHSAR: Prostate epithelial cells (PrECs) immortalized with hTERT, SV40 Large T and small t antigens and overexpressing androgen receptor were grown in PREGM (Lonza CC-3166). HMLE: Human mammary epithelial cells immortalized with hTERT, Large T and small t antigens were grown in MEGM (Lonza CC-3150). All growth media were supplemented with 100 ug/ml Penicillin/Streptomycin. Soft agar colony formation assay The bottom layer contained 0.6% agar (Sigma A5431) in DMEM and 8% FBS. The top layer contained 0.3% agar in PREGM or MEGM for LHSAR or HMLE respectively. Fifteen thousand cells were seeded in the top agar layer in triplicate wells of a 6 well plate. Colonies were counted from 2 to 6 weeks post-seeding. Image of each well was taken at a 66magnification and analyzed with Image J software. Colonies that were 50 sq. pixels or larger were counted. Quantitative RT-PCR Qiagen RNeasy kit was used for RNA extraction. Reverse transcription was carried out with Clonetech Advantage RT-to- PCR kit while the quantitative PCR was carried out using SYBR Green Master Mix (Applied Biosystems). Two sets of NCOA4 and one set of MSMB primers were used: 1) 2) 3) 1) 2) 3) MSMB and NCOA4 Implicated in Prostate Cancer Risk NCOA4 exon 3-4 - Forward CAGCAGCTCTACTCGT- TATTGG Reverse TCTCCAGGCACACAGA- GACT NCOA4 exon 5-6 - Forward CTCTCAAAACCATTCAA- ATTCCT Reverse CTCTGGCATGGAGAT- ACAGC MSMB exon 1-2 - Forward GCTTATCACAATGAATG- TTCTCCT Reverse AATCTCCTGGAACTCCC- TCA PLoS Genetics | www.plosgenetics.org 5 November 2010 | Volume 6 | Issue 11 | e1001204 Expression constructs NCOA4 expression constructs were received from the human ORFeome V5.1 and cloned into pWZL-Neo retroviral expression vector. Retrovirus production, infection and selection were carried out as described previously [41]. RNA interference Short hairpins in pLKO.1 lentiviral constructs were received from the RNAi Consortium (TRC). Lentivirus production and infection were carried out as described previously [42]. Hairpin shMSMB M6 TRC Clone ID TRCN0000147237 Target sequence GTTCTGTCAGTGAATGGATAA Hairpin shMSMB M8 TRC Clone ID TRCN0000146396 Target sequence CACCTTCGTGACTTTATGCAA Hairpin shMSMB M9 TRC Clone ID TRCN0000146343 Target sequence CAAAGGAAACAAACACCCAAT Supporting Information Figure S1 RNA expression of MSMB and NCOA4 in tumor prostate tissue by genotype at rs10994994. Expression in prostate tumor tissue in the Physicians’ Health Study series (n = 59). Each point represents absolute RNA expression for one individual. The top and bottom of the boxes within each graph represent the upper and lower quartiles for expression at each genotype. The band inside each box marks the median value. P-value for association with MSMB expression, 0.0073. P-value for associa- tion with NCOA4 expression, 0.2671. Found at: doi:10.1371/journal.pgen.1001204.s001 (0.50 MB TIF) Figure S2 Expression of TIMM23 at chromosome 10q11 is not associated with genotype at rs10993994 in prostate tissue. A. Expression in histologically normal prostate tissue (n = 84, p = 0.3459). B. Expression in prostate tumor tissue in the Dana- Farber Cancer Institute series (n = 61, p = 0.9939). Found at: doi:10.1371/journal.pgen.1001204.s002 (9.46 MB TIF) Figure S3 MSMB and NCOA4 expression in breast and colon epithelial tissue. Expression of MSMB and NCOA4 isoforms are not consistently associated with genotype at rs10993994 in breast or colon epithelial tissue. A. Expression in histologically normal breast tissue (n = 56). P-values for association between genotype and expression included within each graph. B. Expression in histologically normal colon tissue (n = 72). Found at: doi:10.1371/journal.pgen.1001204.s003 (9.47 MB TIF) Figure S4 Quantitative RT-PCR for the expression of MSMB and NCOA4 in LHSAR cells. A. MSMB suppression with three