Figure 5

ISL-1 promotes the expression of c-Myc in NHL cell lines. (A to B) The expression of ISL-1 and c-Myc were analyzed at both mRNA and protein levels by real-time RT-PCR (A) and Western blot (B) in Raji cells with stable ISL-1 overexpression and Ly3 cells with stable ISL-1 knockdown. (C) Consensus binding site (TAAT box) for ISL-1 on the human c-Myc enhancer was analyzed by MatInspector software. The mutant sequences are presented and they were used to construct mutant c-Myc-luc. (D to E) The transcriptional activity of ISL-1 on c-Myc-luc wide type (D), mutants or deletions (E) was analyzed by luciferase reporter assay in HeLa cells. (“WT”, “M” and “D” represent the plasmid of c-Myc-luc wide type, mutant, or deletion, respectively.). Non, WT and ctrl served as the control in corresponding experiments. (F) ISL-1 recruited on c-Myc promoter was analyzed by ChIP assay. Soluble chromatin was prepared from Ly3 cells followed by immunoprecipitation with the antibody against ISL-1 and the normal IgG served as a control. The DNA extractions were amplified using the primers that covered the ISL-1 binding sites on c-Myc enhancer region by real-time PCR. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01, ^#p < 0.05 vs. the control).