Non-radioactive EMSA was performed on nuclear extracts following the manufacturer’s instructions (Thermo Scientific) using AP1 (59-Biotin-ACGCTTGATGACTCAGCCGGAAT-39 and 59-Biotin-ATTCCGGCTGAGTCATCAAGC-39) and NF-kB (59-Biotin-AAGTTGAGGGGACTTTCCCAGGCT-39 and 59- Biotin-AGCCTGGGAAAGTCCCCTCAACTT-39) oligonucleo- tides. Oct-1 oligonucleotide (5’-Biotin-TGTCGAATGCAAAT- CACTAGAA-3’) was used as a loading control and a 200-fold excess of non-biotinylated oligonucleotides was used as a compe- References 1. O’Brien CA, Lin SC, Bellido T, Manolagas SC (2000) Expression levels of gp130 in bone marrow stromal cells determine the magnitude of osteoclasto- genic signals generated by IL-6-type cytokines. Journal of cellular biochemistry 79: 532–541. 2. Lee JW, Chung HY, Ehrlich LA, Jelinek DF, Callander NS, et al. (2004) IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells. Blood 103: 2308–2315. 3. Bataille R, Chappard D, Klein B (1992) The critical role of interleukin-6, interleukin-1B and macrophage colony-stimulating factor in the pathogenesis of bone lesions in multiple myeloma. International journal of clinical & laboratory research 21: 283–287. 4. Bendre MS, Margulies AG, Walser B, Akel NS, Bhattacharrya S, et al. (2005) Tumor-derived interleukin-8 stimulates osteolysis independent of the receptor activator of nuclear factor-kappaB ligand pathway. Cancer research 65: 11001– 11009. 5. Lisignoli G, Piacentini A, Cristino S, Grassi F, Cavallo C, et al. (2007) CCL20 chemokine induces both osteoblast proliferation and osteoclast differentiation: Increased levels of CCL20 are expressed in subchondral bone tissue of rheumatoid arthritis patients. Journal of cellular physiology 210: 798–806. 6. Gamonal J, Acevedo A, Bascones A, Jorge O, Silva A (2000) Levels of interleukin-1 beta, -8, and -10 and RANTES in gingival crevicular fluid and cell populations in adult periodontitis patients and the effect of periodontal treatment. Journal of periodontology 71: 1535–1545. 7. Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T (2005) Increase of CCL20 expression by human gingival fibroblasts upon stimulation with cytokines and bacterial endotoxin. Clinical and experimental immunology 142: 285–291. 8. Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T (2007) CXC chemokine ligand 16 in periodontal diseases: expression in diseased tissues and production by cytokine-stimulated human gingival fibroblasts. Clinical and experimental immunology 149: 146–154. 9. Choi SJ, Cruz JC, Craig F, Chung H, Devlin RD, et al. (2000) Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma. Blood 96: 671–675. 10. Han JH, Choi SJ, Kurihara N, Koide M, Oba Y, et al. (2001) Macrophage inflammatory protein-1alpha is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor kappaB ligand. Blood 97: 3349–3353. 11. Choi SJ, Oba Y, Gazitt Y, Alsina M, Cruz J, et al. (2001) Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease. The Journal of clinical investigation 108: 1833–1841. 12. Kominsky SL, Abdelmagid SM, Doucet M, Brady K, Weber KL (2008) Macrophage inflammatory protein-1 delta: a novel osteoclast stimulating factor secreted by renal cell carcinoma bone metastasis. Cancer research 68: 1261– 1266. 13. Hotokezaka H, Sakai E, Kanaoka K, Saito K, Matsuo K, et al. (2002) U0126 and PD98059, specific inhibitors of MEK, accelerate differentiation of RAW264.7 cells into osteoclast-like cells. The Journal of biological chemistry 277: 47366–47372. 14. David JP, Sabapathy K, Hoffmann O, Idarraga MH, Wagner EF (2002) JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms. Journal of cell science 115: 4317–4325. MIP-1d Activates NFATc1 via PLCc2 and NFkB titive control for binding specificity. NF-kB and AP-1 activity were evaluated concurrently, thus the same Oct-1 bands appear in Figs. 2D, 3B. Author Contributions Conceived and designed the experiments: KLW MD SLK. Performed the experiments: MD AS NH DH JF SLK. Analyzed the data: KLW MD SLK. Wrote the paper: KLW MD SLK. 15. Mao D, Epple H, Uthgenannt B, Novack DV, Faccio R (2006) PLCgamma2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2. The Journal of clinical investigation 116: 2869–2879. 16. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, et al. (1997) Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nature medicine 3: 1285–1289. 17. Sun SC (2011) Non-canonical NF-kappaB signaling pathway. Cell research 21: 71–85. 18. Yamashita T, Yao Z, Li F, Zhang Q, Badell IR, et al. (2007) NF-kappaB p50 and p52 regulate receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor-induced osteoclast precursor differentiation by activating c-Fos and NFATc1. The Journal of biological chemistry 282: 18245–18253. 19. Maruyama T, Fukushima H, Nakao K, Shin M, Yasuda H, et al. (2010) Processing of the NF-kappa B2 precursor p100 to p52 is critical for RANKL- induced osteoclast differentiation. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 25: 1058–1067. 20. Takayanagi H (2007) Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nature reviews Immunology 7: 292– 304. 21. Matsuo K, Galson DL, Zhao C, Peng L, Laplace C, et al. (2004) Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c- Fos. The Journal of biological chemistry 279: 26475–26480. 22. Vaira S, Johnson T, Hirbe AC, Alhawagri M, Anwisye I, et al. (2008) RelB is the NF-kappaB subunit downstream of NIK responsible for osteoclast differentia- tion. Proceedings of the National Academy of Sciences of the United States of America 105: 3897–3902. 23. Vaira S, Alhawagri M, Anwisye I, Kitaura H, Faccio R, et al. (2008) RelA/p65 promotes osteoclast differentiation by blocking a RANKL-induced apoptotic JNK pathway in mice. The Journal of clinical investigation 118: 2088–2097. 24. Haringman JJ, Smeets TJ, Reinders-Blankert P, Tak PP (2006) Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis. Annals of the rheumatic diseases 65: 294–300. 25. Berahovich RD, Miao Z, Wang Y, Premack B, Howard MC, et al. (2005) Proteolytic activation of alternative CCR1 ligands in inflammation. Journal of immunology 174: 7341–7351. 26. Gladue RP, Brown MF, Zwillich SH (2010) CCR1 antagonists: what have we learned from clinical trials. Current topics in medicinal chemistry 10: 1268– 1277. 27. Uy HL, Dallas M, Calland JW, Boyce BF, Mundy GR, et al. (1995) Use of an in vivo model to determine the effects of interleukin-1 on cells at different stages in the osteoclast lineage. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 10: 295–301. 28. Sanda T, Iida S, Ogura H, Asamitsu K, Murata T, et al. (2005) Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor. Clinical cancer research : an official journal of the American Association for Cancer Research 11: 1974–1982. 29. Victoriano AF, Asamitsu K, Hibi Y, Imai K, Barzaga NG, et al. (2006) Inhibition of human immunodeficiency virus type 1 replication in latently infected cells by a novel IkappaB kinase inhibitor. Antimicrobial agents and chemotherapy 50: 547–555. PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40799