SUPPLEMENTAL TABLE LEGENDS Table S-1: The set of androgen-regulated probes identified by microarray (A) Androgen-induced probes (B) Androgen-repressed probes. The fold-induction or -repression by R1881 is shown.

Table S-2: Androgen-induced ontology biological processes (GOBPs) detected by L2L (A-B) GOBPs significantly enriched in the set of induced by androgen (A) in control cells but not ART- 27-depleted cells (B) in ART-27-depleted cells. (C) All the GOBPs detected in the sets of androgen-induced genes. For each GOBP, the number of probe-sets present on the array (total probes), and the number of genes found in the set of androgen-induced genes detected in control but not ART-27-depleted cells (+ART-27 only), and genes detected in ART-27-depleted cells (-ART-27). Fold-enrichments for each GOBP are also indicated, and GOBPs are significantly enriched if p < 0.01.

SUPPLEMENTAL FIGURE LEGENDS

Figure S-1: Validation of androgen-regulated genes (A-C) LNCaP cells were steroid-deprived for 72 h and stimulated with ethanol vehicle (Veh) or 10 nM R1881 for 17 h. Relative mRNA levels of the indicated androgen-regulated genes (A), checkpoint genes (B) and androgen-insensitive genes (C) were determined by Q-PCR. Data shown is normalized to the ethanol-treated sample, which was arbitrarily set to 1 for each gene. NKX3-1, Nk3 homeobox 1; KRT18, keratin 18; B4GALT1, UDP-Gal:betaGlcNAc beta 1,4- galactosyl transferase, polypeptide 1; FKBP5, FK506 binding protein 5; SORD, sorbitol dehydrogenase; F5, Factor V; SEC24D, SEC24 related gene family, member D; TMEPAI, ; PSA, kallikrein 3 (KLK3)/prostate-specific antigen; PSMD5, (prosome, macropain) 26S subunit, non-ATPase, 5; HPGD, hydroxyprostaglandin dehydrogenase 15-(NAD); KDELR3, KDEL (Lys- Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 3; TNFRSF10B, receptor superfamily, member 10b; ENO2, enolase 2; TLR3, toll-like receptor 3; PRMT1, protein arginine methyltransferase 1; RUVBL1, RuvB-like 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SRC-1; nuclear receptor coactivator 1 (NCOA1)/steroid receptor coactivator 1; IL20RA, interleukin 20 receptor, alpha; SRD5A1, steroid-5-alpha reductase, alpha polypeptide 1; ANG, angiogenin, ITGAV, integrin, alpha V; BMPR1B, bone morphogenetic protein receptor, type 1B; JAG1, jagged 1; ATR, ataxia telangiectasia and Rad3 related; BRIP1, BRCA1-interacting protein C-terminal helicase 1; CCNA2, Cyclin A2; GTSE1, G2 and S-phase expressed 1; CHEK1, Chk1 checkpoint homolog; HUS1, Hydroxyurea sensitive 1 checkpoint homolog; BUB1, Budding uninhibited by benzimidazoles 1 homolog; and CDC6, cell division cycle 6 homolog.

Figure S-2: Stable ART-27-depletion enhances LNCaP cell proliferation (A-B) Pools of LNCaP cells stably expressing a non-silencing shRNA (control) or ART-27-silencing shRNA (shART-27) were steroid-deprived for 3 days, and 30,000 cells were seeded (on day 0), in steroid-free media supplemented with ethanol vehicle (A) or 0.1 nM R1881 (B). The cells were counted after 3 and 5 days using a hemacytometer. (C) Pools of LNCaP cells stably expressing a non-silencing shRNA (control) or ART-27- silencing shRNA (shART-27) were cultured in steroid-free media for 3 days. Relative mRNA levels of the indicated androgen-induced genes were determined by Q-PCR and shown relative to control cells. (D) Pools of LNCaP cells stably expressing a non-silencing shRNA (shControl) or ART-27-silencing shRNA (shART-27) were cultured in steroid-free media supplemented with ethanol vehicle or 0.1 nM R1881 for 3 days. Whole cell extracts were analyzed by western blot.

Figure S-3: Temporal changes in AR and ART-27 recruitment ChIP assay using anti-AR and anti-ART-27 antibodies was performed in LNCaP cells stimulated with 0.1 μM R1881 for the indicated time periods. Recruitment to the indicated regions is shown as a percentage of the input.

SUPPLEMENTAL MATERIALS AND METHODS Stable Reduction of ART-27 Protein in LNCaP cells: ART-27-silencing (AS) shRNAs were designed based on previously validated siRNA sequences. A non-silencing (NS) shRNA that does not match any known human sequence was used as control. The shRNAs were cloned into a pLKO.1 lentiviral vector (described in (1, 2)). The plasmids were retrieved from positive clones by Mini-prep (Qiagen Inc., Valencia, CA) and sequenced. Packaging cells (HEK293-GP) were transiently transfected with 6 µg of lentiviral plasmids and 6 µg of the vesicular stomatitis virus G glycoprotein (VSV-G) plasmid using Fugene reagent (Roche). Media conditioned by the transfectants was collected after 48 and 72 h. Parental LNCaP cells were infected by incubation in conditioned media plus 8 µg/ml Polybrene for 8-12 hours, and allowed to recover for > 12 h. Infected cells were passaged and selected in 1 µg/ml puromycin-treated media for > 6 days. Resistant cells were trypsinized, pooled and expanded, and maintained in RPMI-1640 media supplemented 10% FBS and 1% PS. ChIP assay primer pairs (5’ -3’): NKX3-1ARE: CTGATCAAACGTCACGATGC & GAGGAACAGCTGCTCTCATACA ATR ARE: TGCTGCTCCAGAGACAGATG & CCGCAAAAGGAGATTTGGTA GTSE1 ARE: TTCTGGTCCGTGTTGATCCT & CGTCCGTGTCCTGTCAGATA Q-PCR primers pairs (5’ -3’): ART-27: CAACAGCCTCACCAAGGACT & TCTGCAGGCCTTGTAGTTCTC, and 1 CTGGAGTTGACACTGGCAGA & AGTCCTTGGTGAGGCTGTTG ATR: AGTGAAAGGGCATTCCAAAG & CGCTGCTCAATGTCAAGAAC B4GALT1: GGGAGGAGAAGATGATGACATT & TTGGGCGAGATATAGACATGC BMPR1B: TGAGGGAGATTGTGTGCATC & TGAGTTTTCCCATCTGCCTT BRIP1: GGCCGTCAGTGGTATGAAAT & GAGCTCCCCAATCATTTCTG BUB1: CTCAGCAACAAACCATGGAA & GTGCCAAAGAGCATGCAATA CCNA2: GAATGAGACCCTGCATTTGG & GCCCACAAGCTGAAGTTTTC CDC6: GAGTCAGATGTCAAAAGCCAGAC & AGACCAACCCTCTTGGGAAT CHEK1: TGACTTCCGGCTTTCTAAGG & GGCTGCTCACAATATCAATCAG ENO2: TTGTCAGGGACTATCCTGTGG & CAGCCCAATCATCCTGGT F5: GATCTCTCCAACTCGAGCCTA & AGGGGTGTGGAACATCCATT FKBP5: CGCAGGATATACGCCAACAT & CTTGCCCATTGCTTTATTGG GAPDH: CCTCAACGACCACTTTGTCA & CCCTGTTGCTGTAGCCAAAT GTSE1: CGCAAGAACTCTGCAATGAG & ACATCCACCAGCCTGGAAT HPGD: TGCTTCAAAGCATGGCATAG & CCATTGGCAATCAATGGTG HUS1: AAACACTTTCCCTGCCTCAC & GATGTCATGGGTCACAATGC IL20RA: CACGTTGGCAAAGAGAAACA & TTCAGCAGGCACAAAGAATC ITGAV: GGGATCTCACCCTCAGTGAA & CCAACCTGGCAGACAATCTT JAG1: CGGCCTCTGAAGAACAGAAC & ACCAAGCAACAGATCCAAGC KDELR3: CAAATTGCAGTCGTGTCTGG & CCCTTAAGGACTTTGGTCACA KLK3/ PSA: CCAAGTTCATGCTGTGTGCT & GCACACCATTACAGACAAGTGG KLK4: GTACCACCCCAGCATGTTCT & AGAGTCACCGTTGCAGGAGT KRT18: AGACCCGGGCAGAGAGAC & TCCAGCTTGACCTTGATGTTC NCOR1: CCCTCTTCAACAGGCTCAAC & CAATCGGTGTTGGTGGAGTA NKX3-1: GAGACGCTGGCAGAGACC & TCCAACAGATAAGACCCCAAG, and GAGACGCTGGCAGAGACC & CGCCTGAAGTGTTTTCAGAG PRMT1: AGGAGATCTTCGGCACCAT & GTCGATGGTGAAGTCCAGGT PSMD5: CACTGTGCTGCCTTAAAAGTG & AGTTTCTGAGCCCAGGGTTG RNASE4/ ANG: CGTTTCTGCGGACTTGTTCT & AGCAATGAATGGGTCCTCTG RPL19: CACAAGCTGAAGGCAGACAA & GCGTGCTTCCTTGGTCTTAG RUVBL1: ATTGGCACCAAGACCACACT & CCTTCCCGTTGATTTTAGCA SEC24D: TCCAACAAAAGAGGCCATATTC & TGTCGGAAAACCATTTCTGG SORD: GTGGATATCAAGGGCGTGTT & ACAGACTTGGACGCAAGCAT SRC-1: ACTGCAACCAGCTCTCATCC & GACGTCAGCAAACACCTGAA 2 SRD5A1: ACCAAGGGGAGGCTTATTTG & AGCCACACCACTCCATGATT TLR3: AGGAAAGGCTAGCAGTCATCC & ATCCCAAAGGGCAAAAGG TMEPAI: CCTCGGAGAGCACAGTGTCA & GTAGACCTGCGGCTCTGG TNFRSF10B: CCAGCAAATGAAGGTGATCC & GGAGTCAAAGGGCACCAAG

SUPPLEMENTAL REFERENCES 1. Moffat J, Grueneberg DA, Yang X, et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 2006;124(6):1283-98. 2. Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. Rna 2003;9(4):493-501.

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