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Supplementary Figures S2 and S3 - PDF file 2332K, Figure S2:SG3 tumors are positive for beta-catenin and Myf5 Figure S3:Normal development of Myf5 myoblasts in beta-catenin knockout mice

Supplementary Figure S9 - PDF file 867K,Figure S9: The SYT-SSX2 domain SSXRD executes �-catenin activation and myogenic differentiation

Supplementary Figures S4, S5, S6, S7 - PDF file 1832K, Figure S4:Pyrvinium effect on beta-catenin in SYT-SSX2 expressing C2C12 and synovial sarcoma cells Figure S5:Phosphorylated �-catenin and full-length APC in HS-SY-II and SYO-1 SS cells Figure S6:SSTC-104 effects on growth and beta-catenin in HS-SY-II synovial sarcoma cells Figure S7: LRP6 depletion in HS-SY-II synovial sarcoma cells induces �-catenin nuclear exit

Supplementary Figure S11 and Tables S1-S3 - PDF file 460K, Figure S11: Regulation of an embryonic Wnt-interactive network by SYT-SSX2 and in human synovial sarcomas Table S1: Differentially regulated Wnt components in mesenchymal precursor cells expressing SYT-SSX2 and in synovial sarcoma tumors Table S2: Differentially regulated Wnt targets in mesenchymal precursor cells expressing SYT-SSX2 and in synovial sarcoma tumors Table S3: The embryonic Wnt interactive network in mesenchymal precursor cells expressing SYTSSX2 and in synovial sarcoma tumors

Supplementary Methods and Legends - PDF file 130K, Methods:Additional information on experimental techniques Legends: describing supplementary figures

Cardiac fibrosis is a common sequelae of cardiac injury and has deleterious functional consequences impacting cardiac filling, function and rhythm. Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of tissue fibrosis in mice. To investigate the longitudinal effect of PAI-1 on cardiac structure and function, M-mode echocardiography was employed to examine cardiac function in PAI-1 deficient (PAI-1 −/− ) and wild-type (WT) control mice in four age groups (6,12,18, 24 months). Eighteen month old PAI-1 −/− mice exhibited reduced left ventricular (LV) diastolic internal dimension ( p =0.0118) and a trend towards increased LV posterior wall (LVPW) thickness, compared to WT. Two year old PAI-1 −/− mice showed increased diastolic and systolic LVPW thickness ( p =0.0127 and p =0.0212, respectively), reduced diastolic and systolic LV internal dimension ( p =0.0486 and p =0.0124), but with preserved LV fractional shortening compared to WT. Histological examination of cardiac sections revealed fibrosis on the anterior epicardial surface of the hearts in 18 month old PKO, which in 26 month old mice had become confluent with extensive (10 –17% by area) epicardial, perivascular, and interstitial distribution (compared to none in WT). Real time polymerase chain reaction (RT-PCR) revealed upregulation of transforming growth factor beta (TGF-β) and fibroblast growth factor 2 in PAI-1 −/− compared to WT ( p =0.0234 and p =0.037, respectively). Immunofluoresence confirmed this finding with bright TGF-β staining localized in the media of intra-myocardial arterioles, and phosphorylated SMAD2/3, the downstream TGF-β signaling mediator, in areas of fibrosis. Thoracic aortic cells from aged (18 –24 month) PKO and WT mice were grown in culture, with RT-PCR revealing 4 fold increased TGF-β and 17 fold increased SMAD3 ( p −/− , supplying additional evidence for upregulation of a profibrotic TGF-β/SMAD tissue signaling pathway. The present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways related to aging and PAI-1 deficiency mediated cardiac fibrosis.