Supplementary MaterialsAdditional file 1: Figure S1 ISL-1 is efficiently overexpressed or knockdown in stably transfected NHL cells. further investigation, as these genes contain conserved ISL-1 binding sequences (YTAATGR) on the upstream of the ATG translation start site [16-18]. More importantly, they are remarkably related to the pathogenesis of NHL as previously reported [16-18]. However, the expression of CyclinD1 and BCL-6 did not show a predicted correlation with ISL-1 in NHL cells (data not shown). Therefore, we focused on c-Myc in the rest investigations.Western blot results showed that the basal expression level of c-Myc was positively correlated with the expression level of ISL-1 in NHL cell lines (will be discussed later in Figure? 4A). Moreover, further results indicated that the overexpression of ISL-1 increased the expression of c-Myc C527 at both mRNA and protein levels in Raji cells (Figure? 5A, B left panel). Whereas, the significant decrease of c-Myc expression was associated with the knockdown of ISL-1 as compared with those in the control Ly3 cells (Figure? 5A,B right panel). These results show that ISL-1 could act as a transcriptional activator of c-Myc. Open in a separate window Figure 4 The expression of ISL-1 is positively correlated to the expression of p-STAT3, p-c-Jun and c-Myc. (A) NHL cell lines were analyzed by Western blot with indicated antibodies. (B) Immunohistochemistry for ISL-1, p-STAT3, p-c-Jun and c-Myc expression were performed in multiple specimens of normal lymph nodes (top panel) and NHL patients C527 (bottom panel). Representative images of ISL-1, p-STAT3, p-c-Jun and c-Myc expression levels and cellular distributions in different samples are shown (200 ). Scale bars?=?100?m Open in a separate window 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 C527 to construct mutant wide type (D), mutants or deletions (E) was analyzed by luciferase reporter assay in HeLa cells. (WT, M and D represent the plasmid of 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 Rabbit Polyclonal to CCRL1 were C527 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. values were calculated using a Student (a c-Myc luciferase reporter construct that contains the binding site for ISL-1 on the c-Myc enhancer) showed the stimulated activity in ISL-1-overexpressing cells in a dose-dependent manner, whereas a significant decrease of activity was seen in ISL-1-knockdown cells (Figure? 5D). The constructs containing the mutant or deleted ISL-1 binding site on the c-Myc enhancer (Figure? 5C), M1 (TAAT mutated to cgAT), M2 (TAAT mutated to cggc), D1 (TAAT with TA deleted) and.