were essentially as described [14]. Chromosome paints 4, 7 and 10 were from Euro-Diagnostica (Malm o¨, Sweden). Metaphases were analyzed with an Axioplan 2 Imaging microscope (Carl Zeiss, Oberkochen, Germany) and images were captured using Isis software (MetaSystems, Altiussheim, Germany). Expression plasmids cDNAs of the different ETV1 isoforms were PCR amplified and cloned into pGEM-TEasy (Promega). Inserts were sequence verified and cloned into the pcDNA3 expression vector (Invitro- gen) or the lenti-viral vector pWPXLd (Didier Trono, University of Geneva). Western blot analysis For Western blot analysis, HEK293T cells were transfected with the different pcDNA3-ETV1 expression constructs using the calcium phosphate precipitation method. Cells were harvested 48 h after transfection. Western blot analysis was carried out using standard procedures with antibody directed to the ETV1 C- terminus (Santa Cruz, Santa Cruz, CA, USA). b-actin was used as loading control (Sigma, St Louis, MO, USA). Proteins were visualized by chemiluminescence (Pierce, Rockford, IL, USA). Lentiviral infections HEK293T cells were cotransfected with pWPXLd-ETV1 expression vectors, or pWPXLd-GFP (control), and pPAX2 and pMD2.G (Trono) using the calcium phosphate precipitation method. Virus was harvested from the supernatant and used for infection of PNT2C2 cells. Pools of infected cells were propagated. Soft agar assay A layer of 0.6% low-melting agarose in standard culture medium was prepared in six-well plates. On top, a layer of 0.3% agarose containing 16104 PNT2C2 cells infected with various References 1. Futreal PA, Coin L, Marshall M, Down T, Hubbard T, et al. (2004) A census of human cancer genes. Nat Rev Cancer 4: 177–183. 2. Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, et al. (2005) Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310: 644–648. 3. Hermans KG, van Marion R, van Dekken H, Jenster G, van Weerden WM, et al. (2006) TMPRSS2:ERG fusion by translocation or interstitial deletion is highly relevant in androgen-dependent prostate cancer, but is bypassed in late- stage androgen receptor-negative prostate cancer. Cancer Res 66: 10658–10663. 4. Perner S, Demichelis F, Beroukhim R, Schmidt FH, Mosquera JM, et al. (2006) TMPRSS2-ERG fusion-associated deletions provide insight into the heteroge- neity of prostate cancer. Canser Res 66: 8337–8341. 5. Wang J, Cai Y, Ren C, Ittmann M (2006) Expression of variant TMPRSS2/ ERG fusion messenger RNAs is associated with aggressive prostate cancer. Cancer Res 66: 8347–8351. 6. Tomlins SA, Mehra R, Rhodes DR, Smith LR, Roulstron D, et al. (2006) TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer. Cancer Res 66: 3396–3400. 7. Helgeson BE, Tomlins SA, Shah N, Laxman B, Cao Q, et al. (2008) Characterization of TMPRSS2:ETV5 and SLC45A3:ETV5 gene fusions in prostate cancer. Cancer Res 68: 73–80. ETV1 Translocations in Prostate Cancer ETV1 expressing viruses or control PNT2C2-GFP cells were plated. At day 14, cells were stained with crystal violet and colonies were counted. Supporting Information Figure S1 Part of genomic sequence of ETV1. The different exons are highlighted in yellow. Translation start codons are underlined. Note that the transcript starting at exon 1a may or may not include exon 1a1 but the translation start is the same as that of the transcript starting at exon 1. The end of exon 1b1 is indicated in red. ETV1 transcripts starting at exon 1c lack exons 1, 2 and 3 which results in a truncated TAD in the translated protein. (DOC) Figure S2 Expression of the different ETV1 transcripts was determined by QPCR in the 7900HT Fast Real-Time PCR system from Applied Biosystems using the power SYBR-green master mix (Applied Biosystems). Expression levels are relative to the housekeeping gene PBGD. ETV1 and PBGD primers are listed in Supplementary Table S1. Sample G277 is a BPH. It has very low or no expression of all of the different ETV1 transcripts. Samples G51, G59, G89, G233, G270 and G308 all overexpress ETV1. The different transcripts are expressed in variable levels. (TIF) Table S1 (TIF) Primer sequences. Author Contributions Conceived and designed the experiments: DG HvK HD CdR JT. Performed the experiments: DG HvK HD CdR. Analyzed the data: DG AdK JT. Contributed reagents/materials/analysis tools: HD WMvW. Wrote the paper: DG JT. 8. Tomlins SA, Laxman B, Dhanasekaran SM, Helgeson BE, Cao X, et al. (2007) Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer. Nature 448: 595–599. 9. Attard G, Clark J, Ambroisine L, Mills IG, Fisher G, et al. (2008) Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer. Br J Cancer 99: 314–320. 10. Hermans KG, van der Korput HA, van Marion R, van de Wijngaart DS, Ziel- van der Made A, et al. (2008) Truncated ETV1, fused to novel tissue-specific genes, and full-length ETV1 in prostate cancer. Cancer Res 68: 7541–7549. 11. Wasylyk B, Hahn SL, Giovane A (1993) The Ets family of transcription factors. Eur J Biochem 211: 7–18. 12. Oikawa T, Yamada T (2003) Molecular biology of the Ets family of transcription factors. Gene 303: 11–34. 13. Seth A, Watson DK (2005) ETS transcription factors and their emerging roles in human cancer. Eur J Cancer 41: 2462–2478. 14. Eussen BH, van de Laar I, Douben H, van Kempen L, Hochstenbach R, et al. (2007) A familial inverted duplication 2q33-q34 identified and delinieated by multiple cytogenetic techniques. Eur J Med. Genet 50: 112–119. PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16332