Figure 6

Max is required for Myc repression of the C/EBPδ promoter activity. A. ChIP assays were performed on chromatin isolated from growing (GR) and Growth arrest (GA) HC11 cells using anti-Max antibodies. Immunoprecipitated DNA was amplified using primers flanking the C/EBPδ proximal promoter region (P200) and the C/EBPδ upstream promoter region (P1.8K). "Input" results are derived from PCR amplification of HC11 genomic DNA. Normal rabbit IgG was used as negative control. B. Growing HC11 cells were nucleofected with the "scrambled" siRNA control, Miz1 siRNA and Max siRNA. C/EBPδ promoter-luciferase results were normalized to the Renilla control. The luciferase results for the "scrambled" siRNA control were set as "1". Luciferase results for the Miz1 and Max siRNA treatment groups shown are the average-fold changes relative to the "scrambled" siRNA control values from 3 independent experiments with duplicates performed in each experiment (n = 6). C. HC11 cells were nucleofected with a scrambled siRNA, Max or Miz1 siRNA constructs using the Amaxa nucleofector protocol. Nucleofected HC11 cells were then cultured in complete growth media (proliferating, growing conditions) in the presence of OSM (GROW + OSM). Whole cell lysates were isolated and analyzed by Western blot using anti- C/EBPδ, Max and Miz1 antibodies (lane 1-3). β-actin was assessed as the loading control. The results shown are representative of three independent experiments.