Cell Motility Assays

Biospecimens and associated clinical data related to the study were collected with written consent from the University of Michigan and approved for use by the Internal Review Board. Unbiased metabolomic profiling using liquid/gas-chromatography coupled to mass spectrometry (LC/GC MS) was performed as described 3 (link) using a Thermofisher Linear Ion Trap mass spectrometer with Fourier Transform and Mat-95 XP mass spectrometers respectively (Supplementary Fig.1). Target metabolites were assessed in tissue and urine samples using isotope dilution GC-MS. Metabolomic data analysis is detailed in Supplementary Fig. 4. All Wilcoxon rank-sum tests and t-tests are two-sided using a threshold of P<0.05 for significance. Repeated measures ANOVA is used for the cell line data with p-values from the model F-test. Class-specific metabolomic patterns were visualized using Z-score plots and heat maps. Unsupervised clustering of samples using metabolomic signatures was performed using cluster13 (link) and tree view14 (link) and visualized using heat maps. Network relationship among various molecular concepts and metabolomic data was performed using Oncomine Concept Map4 (link),5 (link)(www.oncomine.org) as outlined in Supplementary Fig. 9. Invasion was measured using a modified Boyden Chamber assay as described10 (link). Cell motility assay was performed as previously reported using blue flourescent microsphere beads15 (link). Targeted knock-down of candidate genes 16 (link) using gene–specific siRNA sequences are listed in Supplementary Table 9. QPCR for enzymes regulating sarcosine levels, EZH2 and ETS were performed as described12 using indicated oligonucleotide primers (Supplementary Table 10). Chromatin immunoprecipitation to interrogate regulatory role of androgen and ETS was performed using published protocols17 (link). ChIP-Seq and digital gene expression were measured using the Genomic DNA sample prep kit and the NIaIII kit on a Genome Analyzer (Illumina) as per manufacturer’s instructions.
Full methods and any associated references are available in the online version of the paper at www.nature.com/nature.

The full-length human Pol η gene codon-optimized for E. coli expression was synthesized by GenScript. The catalytic core (aa 1–432, hPol η) was cloned into modified pET28a45 (link), expressed in E. coli and purified by Ni²⁺-affinity, MonoS and Superdex75 chromatography. The His-tag was removed by PreScission protease. Mutagenesis was performed using QuikChange (Stratagene). Non-hydrolyzable dNMPNPPs were purchased from Jena Bioscience, and phosphoramidites of CPD from Glen Research. CPD oligos were synthesized and purified by TriLink Biotechnogies. Ternary complexes were prepared by mixing WT or C406M mutant hPol η and annealed DNA at a 1:1.05 molar ratio and addition of 5 mM Mg²⁺ and 1 mM non-hydrolyzable deoxynucleotides (dNMPNPP). The final protein concentration was 6–7 mg/ml. Crystals were grown in 0.1 M MES (pH 6.0), 19–21% (w/v) PEG 2K-MME and 5 mM MgCl₂ after several rounds of microseeding. Diffraction data were collected at sectors 22 and 23 of the APS. Phases were determined by molecular replacement46 (link) and multi-wavelength anomalous dispersion using selenomethionine-labeled hPol η47 (link). Structures were refined using CNS48 and interspersed with manual model building using COOT49 . All residues are in the most favorable (97%) and allowed (2.3%) regions of Ramachandran plot except for two that are well defined by electron densities. For functional assays, the C-terminal truncated human Pol η (1–511aa), which has the same TLS activity as the full-length hPol η43 (link), was subcloned into pET21a and readily expressed in E. coli. Q38A and R61A mutations were made using Mutant-K (TaKaRa BIO Inc). Steady-state kinetic assays and primer extension reactions were carried out as described43 (link).