run in TBE £0.5 at room temperature for 2 h at 150 V. The following two (Q/q) 27-bp unmethylated oligonucleotides were used: 5 0 -GATCCTTCGCCTAGGCTC(A/G)CAGCG CGGGAGCGA-3 0 . A methylated q probe (q*) was generated by incorporating a methylated cytosine at the mutated CpG site during oligonucleotide synthesis. Transient transfection assayThe constructs contained 578 bp from IGF2 intron 3 (nucleotides 2868-3446), followed by the IGF2 P3 promoter (nucleotides 2222 to þ45 relative to the start of transcription) 12 and a luciferase reporter. C2C12 myoblast cells were grown to approximately 80% confluence. Cells were transiently co-transfected with the firefly luciferase reporter construct (4 mg) and a Renilla luciferase control vector (phRG-TK, Promega; 80 ng) using 10 mg Lipofectamine 2000 (Invitrogen). Cells were incubated for 25 h before lysis in 100 ml Triton lysis solution. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The results are based on four triplicate experiments using two independent plasmid preparations for each construct. Statistical analysis was done with an analysis of variance.

The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.

Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5 -azacytidine, in part depending on Bmpr1a, a receptor for bone morphogenetic proteins. Given intravenously after ischemia͞reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre͞Lox donor͞ recipient pair (␣MHC-Cre͞R26R), differentiation was shown to occur roughly equally, with and without fusion to host cells. C ardiomyocytes can be formed, at least ex vivo, from diverse adult pluripotent cells (1-5). Apart from therapeutic implications and obviating ethical concerns aroused by embryonic stem cell lines, adult cardiac progenitor cells might provide an explanation distinct from cell cycle reentry, for the reported rare occurrence of cycling ventricular muscle cells (6). However, recent publications suggest the failure of certain stem cells' specification into neurons, skeletal muscle, and myocardium in vivo (7,8) and recommend greater conservatism in evaluating claims of adult stem cell plasticity, for cogent reasons (9-11).The rarity of cardiogenic conversion by endogenous hematopoietic cells (2, 12), requirements for intracardiac injection (3), or mobilization by cytokines (13), uncertain proof for myocytes of host origin in transplanted human hearts (14), and the confounding possibility of cell fusion after grafting in vivo (15, 16) highlight unsettled issues surrounding stem cell plasticity in heart disease. For donor cell types already in clinical studies, the predominant in vivo effect of bone marrow or endothelial progenitor cells may be neoangiogenesis, not cardiac specification (17, 18), and skeletal myoblasts, despite integration and survival, are confounded by arrhythmias, perhaps reflecting lack of transdifferentiation (19). These obstacles underscore the need to seek cardiac progenitor cells beyond the few known sources. Materials and MethodsFlow Cytometry and Magnetic Enrichment. A ''total'' cardiac population was isolated from 6-to 12-wk-old C57BL͞6 mice by coronary perfusion with 0.025% collagenase, as for viable adult mouse cardiomyocytes (20). More typically, a ''myocytedepleted'' population was prepared, incubating minced myocardium in 0.1% collagenase (30 min, 37°C), lethal to most adult mouse cardiomyocytes (20). Cells were then filtered through 70-m mesh. Bone marrow cells (21) were compared, with or without collagenase and filtration. Cells were labeled with stem cell antigen 1 (Sca-1)-phycoerythrin (PE), Sca-1-FITC, c-kit-PE; CD4-...