IL-6 induces ROR1 promoter activity by activating Stat3.

A. A schematic diagram of the ROR1 reporter constructs (left) that were transfected into MM1 cells and maintained in the absence or presence of 20 ng/ml IL-6; their relative luciferase activity is also depicted. The right panel depicts the means ± standard deviation of the relative luciferase activity measured in four different experiments in which transfection efficiency of the truncated ROR1 promoter constructs into MM1 cells was at least 40%. Construct ROR1 −195 and the upstream constructs exhibited significant IL-6-induced luciferase activity. The schematic diagram in the left lower corner depicts four putative Stat3 binding sites. B. Co-transfection efficiency of IL-6-stimulated MM1 cells previously transfected with the ROR +177, ROR1 −195, and ROR1 −360 truncated constructs. C. STAT3-siRNA downregulated the luciferase activity of MM1 cells transfected with ROR1 −195 and ROR1 −360 but not ROR1 +177, suggesting that IL-6 induced luciferase activity by activating Stat3. Student's t test was used to determine whether the differences between luciferase activity values are significant. D. EMSA using biotin-labeled and unlabeled probes of the four putative Stat3 binding sites shows that MM1 cell nuclear protein bound to the biotinylated DNA probes 1 and 3 and that the addition of the corresponding cold (unlabeled) probes attenuated the binding of the hot (labeled) probes. Binding to probe 1 is lighter than probe 3 because of unequal loading. E. Single nucleotide (T to G) mutation either at position −133 or position −418 abrogates the IL-6-induced luciferase activity of ROR1 −834.