JunB promotes Cyp40 transcription in ALK+ ALCL. (A) qRT-PCR analysis of Cyp40 mRNA levels in Karpas 299 and SUP-M2 cells transfected with pooled control or JunB siRNA. (B) Luciferase activity in Karpas 299 cells transfected with a human Cyp40 promoter-driven (pGL2-Cyp40 promoter) or promoter-less (pGL2 basic) luciferase vector, and pooled control or JunB siRNA (left). Results are presented relative to the activity present in cells co-transfected with pGL2-Cyp40 promoter and control siRNA. A western blot demonstrating the level of JunB silencing is shown (right). (C) Luciferase activity in Karpas 299 cells transfected with the luciferase constructs described in (A) along with empty vector (-) or Myc-tagged JunB (+) (top). A western blot illustrating the expression of the Myc-tagged JunB is also included (bottom). (D) Luciferase activity in Karpas 299 cells transfected with the pGL2 basic, pGL2-Cyp40 promoter, or AP-1 mutant pGL2-Cyp40 promoter luciferase construct. Luciferase activity is expressed relative to the activity present in the pGL2-Cyp40 promoter transfected cells. (E) EMSAs were performed using a biotinylated Cyp40 AP-1 probe and no competitor, an unlabeled Cyp40 AP-1 competitor (wt competitor), an unlabeled Cyp40 competitor with a mutation in the AP-1 site (AP-1 mutant competitor) or the anti-JunB or isotype control (control) antibody (Ab). In all experiments, error bars represent the standard deviation of three independent experiments. p values were determined using paired, one-tailed t-tests.