Supporting Information

Li et al. 10.1073/pnas.1200305109 SI Methods treatment, and DHT/Bicalutamide (CDX) treatment were in- Cell Culture, Chemical Treatment, and Functional Assays. Prostate cluded as internal references. Total mapped count in each sample cancer (LNCaP) cells were cultured in RPMI1640 containing was normalized to the median total count of all replicates of DHT 10% (vol/vol) FBS and LNCaP-abl cells in Phenol red-free RPMI and CDX treatments. The Efficacy score (ES) is based on the 1640 containing 10% charcoal/dextran-treated FBS, plus 2 mM contribution of all significantly changed , in which there are N GlutaMax, 10 mM Hepes, 1 mM Sodium Pyruvate, and 2.5 mg/ up-regulated (defined as the fold-change Cd/Cc ≥ 2) and M down- mL D-(+)-Glucose. PC3-control and PC3- regulated (Cd/Cc ≤ 0.5) genes, where Cc and Cd are the average (AR) cells were grown in Phenol red-free RPMI 1640 containing counts of control and DHT treatment, respectively. For i,we 10% charcoal/dextran treated FBS and 0.1 mg/mL Hygromycin denote the range between DHT and control as Cr = jCd – Ccj, and B. AR RNAi and Western blot analysis were as previously de- denote the count from compound treatment as Cm. Using an scribed (1). Cell proliferation and assays were per- androgen up-regulated gene as an example, we denote the con- ’ formed according to the manufacturer s guide (Promega). The tribution esi from gene i in each compound-treated sample. If the AR binding assay was as described (2). compound doesn’t have any effect (i.e., Cm = Cd), we assign esi = For chemical screening, LNCaP cells were first cultured in 0; if it can fully suppress the DHT effect (Cm ≤ Cc), we assign Phenol red-free RPMI1640 medium containing 10% (vol/vol) a maximum positive score esi = 1; if it can even enhance the DHT charcoal/dextran-treated FBS for 2 d. About 3,000 cells were effect (Cm ≥ Cd + Cr), we assign a maximum penalty score esi = seeded in 384-well plates for 1 d followed by treatment with −1. For cases with partial effect, we assign a stepwise score be- chemicals at 5 μM for 2 h before addition of dihydrotestosterone tween −1 and 0 (for androgen agonist effect) or 0 and +1 (an- (DHT) to the final concentration of 50 nM. After 24 h, media ∼ μ μ drogen antagonist effect), where we used step = 10 and esi = were aspirated, leaving 10 L of the media, and 10 L of the stepscore C − C C fi ∑ es N M MELT mixture (Ambion) was added to each well and incubated [( d m)/ r]. The nal ES = i/( + ). The fi at room temperature for 5 min. The plates were kept at −80 °C principle to calculate the Speci city score (SS) is based on the K until the RNA annealing, selection, ligation (RASL) assay. The number ( ) of controls that remain unaltered in compound RASL-seq reaction has recently been detailed (3). treatment. If a compound significantly changed the expression of a control gene j (i.e., Cm/Cc ≥ 2orCm/Cc <= 0.5), we assign a Method for Scoring the Compound Efficacy and Specificity. For each score ssj = 1; otherwise ssj = 0. The integrated specificity score for plate, three replicates of control (DMSO treatment), DHT each compound is SS = 1 −∑ssj/K.

1. Wang D, et al. (2011) Reprogramming transcription by distinct classes of enhancers 3. Li H, Qiu J, Fu X-D (2011) RASL-seq for massive parallel and quantitative analysis of functionally defined by eRNA. Nature 474:390–394. . Curr Protoc Mol Biol, in press. 2. Jones JO, et al. (2009) Non-competitive androgen receptor inhibition in vitro and in vivo. Proc Natl Acad Sci USA 106:7233–7238.

A Correlation between RASL-seq and qPCR B DHT-induced gene expression in detecting DHT-induced gene expression detected by different probes Log2(DHT/Control) RASL-seq log2(DHT/Control)

qPCR log2(DHT/Control) Log2(DHT+Control)

Fig. S1. Validation of high-throughput screening strategy by high-throughput sequencing (HTS2) results by quantitative PCR (qPCR) and fold-changes de- tected by different probe sets that target the same transcripts in LNCaP cells. (A) RT-qPCR validation of a panel of androgen-responsive genes detected by HTS2. (B) A panel of androgen-responsive genes was each targeted by three sets of probes (color-coded as shown). Although different probes showed different targeting efficiencies, they detected similar fold-changes of their target genes in response to DHT treatment.

Li et al. www.pnas.org/cgi/content/short/1200305109 1of5 Fig. S2. Reproducibility and robustness of HTS2.(A) Reproducibility among three sets of biological repeats on mock-treated LNCaP cells. (B) Reproducibility among three sets of biological repeats on DHT-treated LNCaP cells. (C) Pair-wise comparison between mock-treated and DHT-treated LNCaP cells. Red, ex- pected DHT-up-regulated genes; blue, expected DHT-down-regulated genes; green, housekeeping/cytotoxicity genes, which are expected to show little dif- ferences in response to DHT treatment.

Li et al. www.pnas.org/cgi/content/short/1200305109 2of5 Plate 1 Plate 2 Plate 3 Plate 4

Plate 5 Plate 6 Plate 7 Plate 8

Plate 9 Plate 10 Plate 11 Plate 12

Fig. S3. Individual plates from the HTS2 screen. All plates contained internal controls for full-effect mimics (blue dots), DHT treatment (pink dots for lack of effect), and treatments with DHT plus CDX (red dots). Most plates showed anticipated effects for full-effect mimics (high ES and SS), DHT treatment (low ES and high SS), and treatments with DHT and CDX (high ES and SS), except plates 2 and 11 (and to some extent, plates 10 and 12), which exhibited a certain degree of variation.

Li et al. www.pnas.org/cgi/content/short/1200305109 3of5 Table S1. Androgen-induced, cytoxicity, and housekeeping genes Symbol Name Symbol Name

Androgen up-regulated gene Androgen down-regulated gene CHRNA2 Cholinergic receptor, nicotinic, MATN2 Matrilin 2 α-2 (neuronal) TRIM49L1 Tripartite motif containing CCDC83 Coiled-coil domain containing 83 49-like 1 TRPM8 Transient receptor potential OPRK1 Opioid receptor, κ-1 cation channel, subfamily M, member 8 TARP TCR-γ alternate reading UGT2B15 UDP glucuronosyltransferase 2 family, frame polypeptide B15 ORM2 Orosomucoid 2 SI Sucrase-isomaltase (α-glucosidase) F5 Coagulation factor V CAMK2N1 Calcium/calmodulin-dependent protein (proaccelerin, labile factor) kinase II inhibitor 1 KLK2 Kallikrein-related peptidase 2 BCHE Butyrylcholinesterase FAM105A Family with sequence similarity MANEA Mannosidase, endo-α 105, member A MICAL1 Microtubule associated monoxygenase, GRB10 Growth factor receptor-bound protein 10 calponin and LIM domain containing 1 KLK3 Kallikrein-related peptidase 3 OSR2 Odd-skipped related 2 (Drosophila) FKBP5 FK506 binding protein 5 FAM198B Family with sequence similarity 198, member B PMEPA1 Prostate transmembrane protein, DDC Dopa decarboxylase (aromatic androgen induced 1 L-amino acid decarboxylase) C1orf116 1 ORF 116 RFX3 Regulatory factor X, 3 (influences HLA class II expression) SLC45A3 Solute carrier family 45, PKIB Protein kinase (cAMP-dependent, catalytic) member 3 inhibitor-β SGK223 Homolog of rat pragma ATP1B1 ATPase, Na+/K+ transporting, β-1 polypeptide of Rnd2 HERC3 Hect domain and RLD 3 PPP1R14C Protein phosphatase 1, regulatory (inhibitor) subunit 14C EAF2 ELL associated factor 2 SEMA6A Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A ERRFI1 ERBB receptor feedback TMEM38B Transmembrane protein 38B inhibitor 1 NCAPD3 Non-SMC condensin II complex, NCOA7 Nuclear receptor coactivator 7 subunit D3 MBOAT2 Membrane bound FUT8 Fucosyltransferase 8 [α (1,6) O-acyltransferase fucosyltransferase] domain containing 2 ALDH1A3 Aldehyde dehydrogenase SLC12A2 Solute carrier family 12 (sodium/potassium/ 1 family, member A3 chloride transporters), member 2 ELL2 Elongation factor, RNA MANBA Mannosidase, β-A, lysosomal polymerase II, 2 NKX3-1 NK3 homeobox 1 KIF5B Kinesin family member 5B DHCR24 24-Dehydrocholesterol reductase MTF2 Metal response element binding transcription factor 2 PTPRN2 Protein tyrosine phosphatase, SSBP2 Single-stranded DNA binding protein 2 receptor type, N polypeptide 2 RHOU Ras homolog gene family, member U CCDC14 Coiled-coil domain containing 14 ZBTB16 Zinc finger and BTB domain SLC44A1 Solute carrier family 44, member 1 containing 16 LPAR3 Lysophosphatidic acid receptor 3 CXCR7 Chemokine (C-X-C motif) receptor 7 TMPRSS2 Transmembrane protease, serine 2 NIPSNAP3A Nipsnap homolog 3A (Caenorhabditis elegans) MTMR9 Myotubularin related protein 9 Housekeeping gene STK39 Serine threonine kinase 39 ACADVL Acyl-CoA dehydrogenase, very long chain UAP1 UDP-N-acteylglucosamine ATP6V1F ATPase, H+ transporting, lysosomal 14 kDa, pyrophosphorylase 1 V1 subunit F KCNN2 Potassium intermediate/small BCAT2 Branched chain amino acid transaminase 2, conductance calcium-activated mitochondrial channel, subfamily N, member 2

Li et al. www.pnas.org/cgi/content/short/1200305109 4of5 Table S1. Cont. Symbol Name Symbol Name

HOMER2 Homer homolog 2 (Drosophila) CLK1 CDC-like kinase 1 SEPP1 Selenoprotein P, plasma, 1 GPS1 G protein pathway suppressor 1 ACSL3 Acyl-CoA synthetase long-chain IDH3A Isocitrate dehydrogenase 3 (NAD+)-α family member 3 SORD Sorbitol dehydrogenase ILF2 Interleukin enhancer binding factor 2, 45 kDa KCNMA1 Potassium large conductance LAMP1 Lysosomal-associated membrane calcium-activated channel, protein 1 subfamily M, α-member 1 SMS Spermine synthase MYL12A Myosin, light chain 12A, regulatory, nonsarcomeric LDLR Low density lipoprotein receptor NDUFV3 NADH dehydrogenase (ubiquinone) flavoprotein 3, 10 kDa Cytotoxicity gene PDIA6 Protein disulfide isomerase family A, member 6 BAG1 BCL2-associated athanogene PI4KA Phosphatidylinositol 4-kinase, catalytic, α BCL2L1 BCL2-like 1 PSMC1 (prosome, macropain) 26S subunit, ATPase, 1 BCL3 B-cell CLL/lymphoma 3 QARS Glutaminyl-tRNA synthetase CASP2 Caspase 2, apoptosis-related RNPS1 RNA binding protein S1, serine-rich domain cysteine peptidase GSTZ1 Glutathione transferase ζ-1 SEPW1 Selenoprotein W, 1 ILKAP Integrin-linked kinase-associated TALDO1 Transaldolase 1 serine/threonine phosphatase MCM6 Minichromosome maintenance VDAC2 Voltage-dependent anion channel 2 complex component 6 PDCD2 Programmed cell death 2 WARS Tryptophanyl-tRNA synthetase RFC5 Replication factor C (activator 1) 5, 36.5 kDa ACADVL Acyl-CoA dehydrogenase, very long chain XPO1 Exportin 1 (CRM1 homolog, yeast)

Li et al. www.pnas.org/cgi/content/short/1200305109 5of5