Figure 8. Activation of TGFb1/Smad mediated transcription by SF. NIH3T3 cells containing a CAGA12-luc plasmid were treated with TGFb1 alone, TGFb1 and 10 mM DTT, which disrupts the active TGFb1 dimer, or TGFb1 and 2 mM SF. All samples were pre-incubated for 30 minutes and further dialyzed for 4 h so that the final concentration of SF was 34 nM. As an additional negative control cells received no treatment or only 34 nM SF, both of which failed to induce luciferase. Chemiluminescence was normalized to the protein concentration of each sample (for details see Methods). This is a representative experiment of a total of four similar experiments performed. Data shown are mean (s.e.m) of three replicates. doi:10.1371/journal.pone.0002568.g008 Table S2 Relative expression of probes belonging to the TGFb receptor pathway in GSTM1 positive individuals compared with GSTM1 nulls following six months broccoli-rich diet (P#0.05). Found at: doi:10.1371/journal.pone.0002568.s002 (0.10 MB DOC) Table S3 Relative expression of probes belonging to the EGF receptor pathway in GSTM1 positive individuals compared with GSTM1 nulls following six months broccoli-rich diet (P#0.05). Found at: doi:10.1371/journal.pone.0002568.s003 (0.12 MB DOC) Table S4 Change in expression of probes of the TGFb receptor pathway in paired samples before and after a 12 month broccoli- rich diet. References 1. Fleshner N, Zlotta AR (2007) Prostate cancer prevention: past, present, and future. Cancer 110: 1889–1899. 2. Kristal AR, Lampe JW (2002) Brassica vegetables and prostate cancer risk: a review of the epidemiological evidence. Nutr Cancer 42: 1–9. 3. Lin HJ, Probst-Hensch NM, Louie AD, Kau IH, Witte JS, et al. (1998) Glutathione transferase genotype, broccoli, and lower prevalence of colorectal adenomas. Cancer Epidemiol Biomarkers Prev 7: 647– 652. 4. Spitz MR, Duphorne CM, Detry MA, Pillow PC, Amos CI, et al. (2000) Dietary intake of isothiocyanates: evidence of a joint effect with glutathione S-transferase polymorphisms in lung cancer risk. Cancer Epidemiol Biomarkers Prev 9: 1017–1020. 5. Wang LI, Giovannucci EL, Hunter D, Neuberg D, Su L, et al. (2004) Dietary intake of Cruciferous vegetables, Glutathione S-transferase (GST) polymor- phisms and lung cancer risk in a Caucasian population. Cancer Causes Control 15: 977–985. 6. Ambrosone CB, McCann SE, Freudenheim JL, Marshall JR, Zhang Y, et al. (2004) Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. J Nutr 134: 1134–1138. 7. Fowke JH, Chung FL, Jin F, Qi D, Cai Q, et al. (2003) Urinary isothiocyanate levels, brassica, and human breast cancer. Cancer Res 63: 3980–3986. Broccoli & Signalling Pathways Found DOC) at: doi:10.1371/journal.pone.0002568.s004 (0.08 MB Text S1 Found DOC) Table S5 Change in expression of probes of the EGF receptor pathway in paired samples before and after a 12 month broccoli- rich diet. Found at: doi:10.1371/journal.pone.0002568.s005 (0.09 MB DOC) Protocol S1 Trial Protocol. Found at: doi:10.1371/journal.pone.0002568.s006 (0.31 DOC) Checklist S1 CONSORT Checklist. Found at: doi:10.1371/journal.pone.0002568.s007 (0.07 DOC) Approval from Norwich Research Ethics Committee. at: doi:10.1371/journal.pone.0002568.s008 (0.07 MB Figure S1 CONSORT flow diagram. Found at: doi:10.1371/journal.pone.0002568.s009 (0.03 DOC) Figure S2 Modification of TGFb1 by SF and iodoacetamide. Found at: doi:10.1371/journal.pone.0002568.s010 (0.07 MB DOC) Acknowledgments We would like to thank Dr. Jack Dainty for advice on statistical analyses. We are grateful to Iain Sheriffs of the Norfolk & Norwich University Hospital Partners in Cancer Research Human Tissue Bank for help with tissue banking, Zoe Emmerson and Henrik Townsend of the Nottingham Arabidopsis Stock Centre at the University of Nottingham for processing arrays, Charlotte Sayce and the staff of the Human Nutrition Unit, Institute of Food Research for assistance with the intervention study, and Shikha Saha of the Institute of Food Research for assistance with biochemical analyses. We would also like to thank Martyn Watling (ADAS, Terrington, UK) for assistance with broccoli cultivation and Sarah Pettitt (Christian Salvensen) for broccoli processing. We are also grateful to Nathalie Juge for critical review of the manuscript. Author Contributions Conceived and designed the experiments: RM MT. Performed the experiments: MT AG AM JB VF AJ CO. Analyzed the data: DB RM MT AG AM PN VF AC AJ CO RB RM. Contributed reagents/materials/ analysis tools: AC. Wrote the paper: RM MT. MB MB MB 8. van Poppel G, Verhoeven DT, Verhagen H, Goldbohm RA (1999) Brassica vegetables and cancer prevention. Epidemiology and mechanisms. Adv Exp Med Biol 472: 159–168. 9. Moore LE, Brennan P, Karami S, Hung RJ, Hsu C, et al. (2007) Glutathione S- transferase polymorphisms, cruciferous vegetable intake and cancer risk in the Central and Eastern European Kidney Cancer Study. Carcinogenesis 28: 1960–1964. 10. Cornelis MC, El-Sohemy A, Campos H (2007) GSTT1 genotype modifies the association between cruciferous vegetable intake and the risk of myocardial infarction. Am J Clin Nutr 86: 752–758. 11. Joseph MA, Moysich KB, Freudenheim JL, Shields PG, Bowman ED, et al. (2004) Cruciferous vegetables, genetic polymorphisms in glutathione S- transferases M1 and T1, and prostate cancer risk. Nutr Cancer 50: 206–213. 12. Kirsh VA, Peters U, Mayne ST, Subar AF, Chatterjee N, et al. (2007) Prospective study of fruit and vegetable intake and risk of prostate cancer. J Natl Cancer Inst 99: 1200–1209. 13. Conaway CC, Getahun SM, Liebes LL, Pusateri DJ, Topham DKW, et al. (2000) Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli. Nutrition and Cancer-an International Journal 38: 168–178. 14. Gasper AV, Al-Janobi A, Smith JA, Bacon JR, Fortun P, et al. (2005) Glutathione S-transferase M1 polymorphism and metabolism of sulforaphane from standard and high-glucosinolate broccoli. Am J Clin Nutr 82: 1283–1291. PLoS ONE | www.plosone.org 13 July 2008 | Volume 3 | Issue 7 | e2568