Figure 2
Reptin cooperates with c-MYC to regulate the hTERT promoter activity. (A and B) c-MYC-dependent inhibition of the hTERT promoter activity by Reptin depletion. AGS and HGC-27 cells were first treated with control (C), Reptin (R) or Pontin (P) siRNA and then transfected with either p181wt or its c-MYC mutant variant p181MYC-. Luciferase activity was assessed using a dual luciferase detection kit 48 hours after transfection and relative luciferase activity was obtained from its normalization by co-transfected thymidine kinase renilla activity and expressed as percentage of that in control cells. Bars: Standard deviation. * and **: P < 0.05 and 0.01, respectively. Bars: Standard deviations. NS: Not significant. Three independent experiments were performed. (C) Concomitant presence or absence of Reptin and c-MYC on the hTERT promoter. AGS and HGC-27 cells were treated with control or Reptin siRNA and chromatin immunoprecipitation was then performed to examine the association between the hTERT promoter sequence and Reptin, c-MYC, or acetylated histone H3. (D) c-MYC expression in Reptin-depleted gastric cancer cells as determined by using rqPCR (Left panel) and western blot (Right panel). NS: Not significant and **: P < 0.01. (E) Physical interaction between Reptin and c-MYC as demonstrated using immunoprecipitation. Cellular proteins derived from AGS and BGC-823 cells were precipitated by a c-MYC antibody followed by detection using the antibody against Reptin. Rabbit IgG was used as a negative control. Two independent experiments were performed with similar results.