Supplementary Figure 1. Manhattan plot of the Discovery stage. SNPs in the NFIB risk locus are highlighted in green. Supplementary Figure 2. LD structure of the NFIB risk locus. Plot was made using the CEU population of the 1000 Genomes Project data with LDlink (ref 54). Supplementary Figure 3. Significant relationship between the genotype of the metastasis associated SNP rs7034162, and expression of NFIB and IGFBP5 in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of NFIBbetween the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 4. No significant relationship between the genotype of the metastasis associated SNP, rs7034162, and expression of protein-encoding genes FREM1, ZDHHC1, and MPDZ in the neighborhood of NFIB in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of FREM1, ZDHHC1, and MPDZ between the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 5. The human osteosarcoma cell lines, U2OS and HOS, had higher NFIB protein levels then OSA cells. NFIB levels were determined using immunoblot analysis in the OSA, HOS and U2OS cells both basal (A) and after treatment with siRNA against NFIB (B). Supplementary Figure 6. NFIB expression significantly correlates with IGFBP5 expression in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the expression levels of NFIB with the corresponding IGFBP5 expression levels. Supplementary Figure 7. Expression levels of IGFBP5 decreases after NFIB suppression of human osteosarcoma cell lines. IGFBP5 expression of OSA, HOS and U2OS cells was determined 48 hours after transfection with control siRNA (si-NEG) or siRNA targeting NFIB (si-NFIB). Graph showing relative expression compared to control treated U2OS cells. Supplementary Figure 8. A model of how the NFIB risk locus leads to changes in NFIB expression and potentially may affect osteosarcoma metastatic potential. The risk allele (right panel) leads to lowered expression of NFIB and NFIB-mediated lower expression of IGFPB5, which then leads to less IGFBP5-mediated inhibition of IGF-1 (right panel, grey arrows), leading to an increase in proliferation, survival and metastasis of osteosarcoma cells (right panel, black arrows). The reference allele (left panel) leads to both higher expression of NFIB and higher expression of NFIB-mediated IGFPB5. In turn, higher IGFBP5 levels leads to increased inhibition of IGF-1 (left panel, black arrows), leading to a decrease in proliferation, survival and metastasis of osteosarcoma cells (left panel, grey arrows). Supplementary Figure 9. Plot of the PCA Eigenvectors of the Discovery stage. Supplementary Figure 10. QQ-plot of the Discovery stage.