Found at: doi:10.1371/journal.pone.0012996.s001 (0.16 PDF) MB Dataset S1 Significant transcriptional changes caused by the compounds (5926 transcripts, P,0.01). Found at: doi:10.1371/journal.pone.0012996.s002 (3.03 MB XLS) Dataset S2 Genes regulated by all compounds (118 genes, P,0.01). Found at: doi:10.1371/journal.pone.0012996.s003 (0.08 MB XLS) Figure S1 qPCR analysis of TP53 mRNA expression. MCF-7 cells were incubated with BMH-7, -9 (10 mM), BMH-15, -22, -23 (5 mM), and BMH-21 (0.5 mM) for 6 h, and control cultures were mock-treated with DMSO (n = 4). Total RNA was isolated and qPCR performed for TP53. The values were normalized according to GAPDH. Error bars represent SD. Found at: doi:10.1371/journal.pone.0012996.s004 (0.15 MB PDF) Figure S2 p53 dependency of cell viability. Lead compound effect on p53 isogenic HCT116 cells. HCT116 p53+/+ and p532/2 cells were cultured in the presence of BMH-7, -9, -15, -22, -23 (5 mM), BMH-21 (0.5 mM), and nutlin-3 (5 mM) for 72 h followed by counting of the cells. The relative viability response as adjusted to controls in the p53+/+ as compared to p532/2 cells is shown. Error bars represent SE. Found at: doi:10.1371/journal.pone.0012996.s005 (0.16 MB PDF) Figure S3 Human ex vivo prostate tissue. Fresh prostate tissues were obtained from radical prostatectomies, and the sections were incubated with BMH-7 (20 mM), BMH-9 (20 mM), BMH-15 (20 mM), BMH-21 (2 mM), and BMH-22 (20 mM) for 24 h, fixed and stained for p53 and DNA, and in (B) also for gamma-H2AX. Images were captured using confocal microscopy (A) or wide-field microscopy (B). Prostate glands are indicated by white dashed lines. Scale bar, 50 mm. Found at: doi:10.1371/journal.pone.0012996.s006 (0.53 MB PDF) Figure S4 In vivo toxicity. Mice were injected intraperitoneally with BMH-7, -9, -15, -22 (20 mg/kg), and BMH-21 (2 mg/kg) three times a week for three weeks in 30 ml DMSO. Control animals received only the DMSO vehicle. The mice were sacrificed and organs (thymus, spleen, intestine, liver and kidney) were collected for histological examination. No acute or chronic toxicities were observed based on the histological hematoxylin- eosin analyses. Similarly, there were no changes in the weight curves of mice undergoing the above treatment regimen (N = 2 for each treatment group) (data not shown). Found at: doi:10.1371/journal.pone.0012996.s007 (1.16 MB PDF) Figure S5 DNA unwinding. TOP1 (2 U) was added to plasmid DNA to allow full relaxation of the plasmid (Rel). Subsequently, an References 1. Kastan MB (2007) Wild-type p53: tumors can’t stand it. Cell 128: 837–40. 2. Vousden KH, Prives C (2009) Blinded by the light: The growing complexity of p53. Cell 137: 413–31. 3. Vousden KH, Lane DP (2007) p53 in health and disease. Nat Rev Mol Cell Biol 8: 275–83. 4. Soussi T, Be´roud C (2001) Assessing TP53 status in human tumours to evaluate clinical outcome. Nat Rev Cancer 1: 233–40. 5. Levesque AA, Eastman A (2007) p53-based cancer therapies: Is defective p53 the Achilles heel of the tumor? Carcinogenesis 28: 13–20. Novel p53 Pathway Activators excess of TOP1 (20 U) and increasing amounts of compounds (BMH-7, -9, -15, -22, -23, 0.01-5 mM; BMH-21, 0.001-0.5 mM) were added and incubated for further 1 h at 37uC. The reaction was quenched and the samples were analyzed by agarose gel electrophoresis. Note appearance of DNA topomers (Rn) and supercoiled DNA (Sc) due to intercalation. D, DMSO control, EB ethidium bromide. Found at: doi:10.1371/journal.pone.0012996.s008 (0.22 MB PDF) Table S1 p53 activating drugs in the Spectrum Collection. Found at: doi:10.1371/journal.pone.0012996.s009 (0.06 PDF) Table S2 Lipinsky rule of five. Found at: doi:10.1371/journal.pone.0012996.s010 (0.06 PDF) Table S3 In vitro normal and melanoma cell line viability responses. Found at: doi:10.1371/journal.pone.0012996.s011 (0.07 MB PDF) Table S4 DNA intercalation of the compounds by UV-VIS. Found at: doi:10.1371/journal.pone.0012996.s012 (0.06 MB PDF) Table S5 GO categories of transcriptional targets. Found at: doi:10.1371/journal.pone.0012996.s013 (0.06 PDF) Table S6 KEGG pathways of transcriptional targets. Found at: doi:10.1371/journal.pone.0012996.s014 (0.07 PDF) Table S7 Summary of top-ranking connectivities. Found at: doi:10.1371/journal.pone.0012996.s015 (0.06 PDF) MB MB MB MB MB Acknowledgments We thank K. Alitalo, T. DeWeese, and B. Nelson for critical comments, M. Salo, A-H. Sukupolvi, and S. Hao for technical assistance, H. Hao for help with statistical analyses, J. Stivers for advice and reagents on TOP1 and DNA unwinding assay, O. Saksela for primary melanocytes, P. Ojala and E. Cesarman for BC-3 cells and derivative, T. Petrova for HIMEC cells, A. Sankila, L. Andersson and M. Ruutu for help with prostate specimens, Biomedicum High Throughput Center, Meilahti Experimental Animal Center, Johns Hopkins Microchip Center for assistance, and Laiho labs both at University of Helsinki and Johns Hopkins University for discussions and critical comments. Author Contributions Conceived and designed the experiments: KP LC HL PL ML. Performed the experiments: KP LC HL SJ HMM JE PL AV RJJ TMaH. Analyzed the data: KP LC HL SJ HMM PL RJJ TMaH ML. Contributed reagents/ materials/analysis tools: ML. Wrote the paper: KP LC ML. 6. Malkin D (1994) Germline p53 mutations and heritable cancer. Annu Rev Genet 28: 443–65. 7. Toledo F, Wahl GM (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6: 909–23. 8. Martins CP, Brown-Swigart L, Evan GI (2006) Modeling the therapeutic efficacy of p53 restoration in tumors. Cell 127: 1323–34. 9. Ventura A, Kirsch DG, McLaughlin ME, Tuveson DA, Grimm J, et al. (2007) Restoration of p53 function leads to tumour regression in vivo. Nature 445: 661–5. PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12996