Introduction 1 2 8 9 10 11 12 13 14 ICAD 15 ICAD ICAD ICAD 16 ICAD ICAD Materials and methods Cell cultures 2 ICAD ICAD ICAD EcoR 5′ rapid amplification of cDNA ends (5′-RACE) assay ICAD + ICAD ICAD Plasmid constructions ICAD Sac Hind ICAD Sac Hind Transfection and reporter assay 4 2 ICAD 17 Chromatin immunoprecipitation Chromatin immunoprecipitation assays were performed using the ChIP assay kit (Upstate). Briefly, cells in 100-mm dishes were grown to 70% confluency over 48 h. The chromatin from formaldehyde-fixed cells was sonicated and immunoprecipitated using mouse monoclonal anti-c-Myc or anti-N-Myc antibodies (Santa Cruz). The chromatin immunoprecipitate was analysed by PCR with the following primer pairs: F1 (5′-CGAGCTCGGTATACATGCGTGTGCATCG-3′) and R1 (5′-CAAGCTTGCCTCCACAAGGTGGGACCTG-3′) for amplifying the region from nt −272 to −71 containing potential Myc-binding sites; and F2 (5′-GAGATCAAAACTGCAGTGAG-3′) and R2 (5′-CACTGTTGGAGATTGTTCAG-3′) for amplifying the region from nt −789 to −451 that does not contain Myc-binding sites. Western blotting Cells were washed with PBS and lysed in SDS sample buffer. Cell lysate samples were separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Immobilion; Millipore). After blocking in nonfat milk solution (Blocking One; Nakaraitesk), the membranes were probed with monoclonal antibody against the ICAD protein (Santa Cruz), c-Myc (Sigma), or N-Myc (Santa Cruz), as the primary antibody for 1 h. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin as the secondary antibody, followed by visualization with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). siRNA experiments c-Myc, N-Myc c-Myc N-Myc RNA preparation and real-time quantitative RT-PCR Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNAs were synthesized using the SuperScriptIII First-Strand Synthesis kit (Invitrogen) and then used as templates for real-time PCR. Quantitative PCR was performed using the ABI Prism 7700 sequence detection system using Taq-Man Gene Expression assays (Applied Biosystems). The standard curve was created using serially diluted total RNA obtained from Huh-7 cultures and β-actin was chosen as the internal standard to control for variability in amplification. Amplification was performed at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 60 s. Results and discussion ICAD ICAD 1 1 ICAD 18 ICAD Fig. 1 a b ICAD  ICAD ICAD ICAD 19 ICAD http://cpgislands.usc.edu/ ICAD 1 ICAD 2 2 ICAD Fig. 2 ICAD ICAD n  ICAD 1 20 23 ICAD 24 25 26 27 3 Fig. 3 a ICAD n b ICAD  ICAD 3 ICAD ICAD ICAD ICAD ICAD ICAD ICAD 4 16 N-Myc 28 ICAD 4 ICAD ICAD Fig. 4 a ICAD ICAD b ICAD  Myc-dependent expression of the ICAD protein ICAD 5 5 Fig. 5 a b c c-Myc ICAD n  ICAD Myc ICAD c-Myc ICAD 5 ICAD c-Myc 11 14 29 30 ICAD ICAD ICAD ICAD 25 31 32 c-myc N-myc 25 33 34 32 35 36 37 38 ICAD ICAD 16 ICAD ICAD 16 ICAD ICAD 39 ICAD