Supplementary Figures 1 - 11 from A Genome-Wide Scan Identifies Variants in <i>NFIB</i> Associated with Metastasis in Patients with Osteosarcoma
Authors: Mirabello Lisa , Koster Roelof , Moriarity Branden S. , Spector Logan G. , Meltzer Paul S. , Gary Joy , Machiela Mitchell J. , Pankratz Nathan , Panagiotou Orestis A. , Largaespada David , Wang Zhaoming , Gastier-Foster Julie M. , Gorlick Richard , Khanna Chand , de Toledo Silvia Regina Caminada , Petrilli Antonio S. , Patiño-Garcia Ana , Sierrasesúmaga Luis , Lecanda Fernando , Andrulis Irene L. , Wunder Jay S. , Gokgoz Nalan , Serra Massimo , Hattinger Claudia , Picci Piero , Scotlandi Katia , Flanagan Adrienne M. , Tirabosco Roberto , Amary Maria Fernanda , Halai Dina , Ballinger Mandy L. , Thomas David M. , Davis Sean , Barkauskas Donald A. , Marina Neyssa , Helman Lee , Otto George M. , Becklin Kelsie L. , Wolf Natalie K. , Weg Madison T. , Tucker Margaret , Wacholder Sholom , Fraumeni Joseph F. , Caporaso Neil E. , Boland Joseph F. , Hicks Belynda D. , Vogt Aurelie , Burdett Laurie , Yeager Meredith , Hoover Robert N. , Chanock Stephen J. , Savage Sharon A. 
Published: April 2023

<p>Supplementary Figure 1. Manhattan plot of the Discovery stage. SNPs in the <i>NFIB</i> risk locus are highlighted in green. Supplementary Figure 2. LD structure of the <i>NFIB</i> 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 <i>NFIB</i> and <i>IGFBP5</i> in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of <i>NFIB</i>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 4. No significant relationship between the genotype of the metastasis associated SNP, rs7034162, and expression of protein-encoding genes <i>FREM1</i>, <i>ZDHHC1</i>, and <i>MPDZ</i> in the neighborhood of <i>NFIB</i> in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of <i>FREM1</i>, <i>ZDHHC1</i>, and <i>MPDZ</i> 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 <i>NFIB</i> (B). Supplementary Figure 6. <i>NFIB</i> expression significantly correlates with <i>IGFBP5</i> expression in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the expression levels of <i>NFIB</i> with the corresponding <i>IGFBP5</i> expression levels. Supplementary Figure 7. Expression levels of IGFBP5 decreases after <i>NFIB</i> suppression of human osteosarcoma cell lines. <i>IGFBP5</i> 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 <i>NFIB</i> 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.</p>