Supporting Information Figure S1 Generation of BE(2)-C-derived cell lines with in- ducible expression of individual HOXD genes. (A) qRT-PCR analysis of HOXD mRNA levels in BE(2)-C/Tet-Off/myc-HOXD cells cultured in presence (Doxy+, 2 mg/ml) or absence of doxycycline (Doxy-) for 3 days. The HOXD mRNA levels in the presence of Doxy were designated as 1.0. The data were from two independent samples with each being assayed in triplicates. Error bars, SD. (B) Immunoblot analysis of myc-HOXD proteins in the absence of Doxy for 3 or 6 days. Beta-actin levels are shown as loading control. (TIF) Figure S2 Induction of HOXD genes has no effect on the expression of cyclin D1, CDK4 and CDK6. Immunoblot analysis of BE(2)-C/Tet-Off/myc-HOXD cells cultured in the presence (2 mg/ml) or absence of Doxy for 3 or 6 days (the same cell samples shown in Figure 3). Alpha-tubulin levels are shown as loading control. The data are representative of two independent experiments with similar results. (TIF) Figure S3 HOXD8 is a mediator of RA action. (A) qRT-PCR analysis of HOXD8 mRNA levels in BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/HOXD8sh-2 cells in the presence (2 mg/ ml) or absence of Doxy for 6 days. The HOXD8 mRNA level in BE(2)-C/Tet-Off/luc-sh cells in the presence of Doxy was designated as 1.0. The induction of HOXD8sh-2 resulted in an average of 68% reduction in HOXD8 mRNA levels. The data were from two independent samples with each being assayed in triplicate and analyzed using two-tailed Student’s t-test with the p values indicated. Error bars, SD. (B) FACS analysis of the cell cycle status of BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/ References 1. Alexander T, Nolte C, Krumlauf R (2009) Hox genes and segmentation of the hindbrain and axial skeleton. Annu Rev Cell Dev Biol 25: 431–456. 2. Maden M (2007) Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat Rev Neurosci 8: 755–765. 3. Pearson JC, Lemons D, McGinnis W (2005) Modulating Hox gene functions during animal body patterning. Nat Rev Genet 6: 893–904. 4. Moens CB, Selleri L (2006) Hox cofactors in vertebrate development. Dev Biol 291: 193–206. 5. Shah N, Sukumar S (2010) The Hox genes and their roles in oncogenesis. Nat Rev Cancer 10: 361–371. 6. Jones-Villeneuve EM, McBurney MW, Rogers KA, Kalnins VI (1982) Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells. J Cell Biol 94: 253–262. 7. McBurney MW, Jones-Villeneuve EM, Edwards MK, Anderson PJ (1982) Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature 299: 165–167. 8. Strubing C, Ahnert-Hilger G, Shan J, Wiedenmann B, Hescheler J, et al. (1995) Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech Dev 53: 275– 287. 9. Sidell N (1982) Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. J Natl Cancer Inst 68: 589–596. 10. Mavilio F, Simeone A, Boncinelli E, Andrews PW (1988) Activation of four homeobox gene clusters in human embryonal carcinoma cells induced to differentiate by retinoic acid. Differentiation 37: 73–79. 11. Simeone A, Acampora D, Arcioni L, Andrews PW, Boncinelli E, et al. (1990) Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature 346: 763–766. 12. Simeone A, Acampora D, Nigro V, Faiella A, D’Esposito M, et al. (1991) Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mech Dev 33: 215–227. 13. Peverali FA, D’Esposito M, Acampora D, Bunone G, Negri M, et al. (1990) Expression of HOX homeogenes in human neuroblastoma cell culture lines. Differentiation 45: 61–69. 14. Manohar CF, Furtado MR, Salwen HR, Cohn SL (1993) Hox gene expression in differentiating human neuroblastoma cells. Biochem Mol Biol Int 30: 733– 741. HOXD Regulation of Neuroblastoma Differentiation HOXD8sh-2 cells that were cultured in the absence of Doxy for 6 days and then either untreated or treated with 10 mM RA for 3 or 6 days. Shown are representatives of two independent experiments with similar results. (TIF) Figure S4 HOXD9 is not required for RA action. (A) qRT-PCR analysis of HOXD9 mRNA levels in BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/HOXD9-sh cells cultured in the presence (2 mg/ml) or absence of Doxy for 6 days. The HOXD9 mRNA level in BE(2)-C/Tet-Off/Luc-sh cells in the presence of Doxy was designated as 1.0. The induction of HOXD9-sh resulted in an average of 69% reduction in HOXD9 mRNA levels. The data were from two independent samples with each being assayed in triplicate and analyzed using two-tailed Student’s t-test with the p values indicated. (B–D) Phase contrast imaging (B) and cell cycle analysis (C–D) of BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/ HOXD9-sh cells that were cultured in the absence of Doxy for 6 days and then either untreated or treated with 10 mM RA for 3 or 6 days. The data presented in (D) were from three independent experiments and analyzed using two-tailed Student’s t-test with the p values indicated. Error bars (A and D), SD. (TIF) Table S1 (DOC) Table S2 (DOC) qRT-PCR primers. ChIP-qPCR primers. Author Contributions Conceived and designed the experiments: YZ HFD. Performed the experiments: YZ ED LY LM XW BAM. Analyzed the data: YZ ED SH HFD XW. Wrote the paper: YZ HFD. 15. Manohar CF, Salwen HR, Furtado MR, Cohn SL (1996) Up-regulation of HOXC6, HOXD1, and HOXD8 homeobox gene expression in human neuroblastoma cells following chemical induction of differentiation. Tumour Biol 17: 34–47. 16. Brodeur GM (2003) Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3: 203–216. 17. Maris JM (2010) Recent Advances in Neuroblastoma. New England Journal of Medicine 362: 2202–2211. 18. Beckwith JB, Martin RF (1968) Observations on the histopathology of neuroblastomas. J Pediatr Surg 3: 106–110. 19. Hughes M, Marsden HB, Palmer MK (1974) Histologic patterns of neuroblastoma related to prognosis and clinical staging. Cancer 34: 1706–1711. 20. Shimada H, Chatten J, Newton WA Jr., Sachs N, Hamoudi AB, et al. (1984) Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. J Natl Cancer Inst 73: 405–416. 21. Ambros IM, Hata J, Joshi VV, Roald B, Dehner LP, et al. (2002) Morphologic features of neuroblastoma (Schwannian stroma-poor tumors) in clinically favorable and unfavorable groups. Cancer 94: 1574–1583. 22. Cohn SL, Pearson ADJ, London WB, Monclair T, Ambros PF, et al. (2009) The International Neuroblastoma Risk Group (INRG) Classification System: An INRG Task Force Report. Journal of Clinical Oncology 27: 289–297. 23. Reynolds CP, Matthay KK, Villablanca JG, Maurer BJ (2003) Retinoid therapy of high-risk neuroblastoma. Cancer Lett 197: 185–192. 24. Volchenboum SL, Cohn SL (2009) Progress in defining and treating high-risk neuroblastoma: lessons from the bench and bedside. J Clin Oncol 27: 1003– 1004. 25. Mao L, Ding J, Zha Y, Yang L, McCarthy BA, et al. (2011) HOXC9 Links Cell- Cycle Exit and Neuronal Differentiation and Is a Prognostic Marker in Neuroblastoma. Cancer Res 71: 4314–4324. 26. Ross RA, Spengler BA, Domenech C, Porubcin M, Rettig WJ, et al. (1995) Human neuroblastoma I-type cells are malignant neural crest stem cells. Cell Growth Differ 6: 449–456. 27. Cui H, Ma J, Ding J, Li T, Alam G, et al. (2006) Bmi-1 regulates the differentiation and clonogenic self-renewal of I-type neuroblastoma cells in a concentration-dependent manner. J Biol Chem 281: 34696–34704. 28. Boncinelli E, Simeone A, Acampora D, Mavilio F (1991) HOX gene activation by retinoic acid. Trends Genet 7: 329–334. PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e40728