Figure 6. VentX transactivates M-CSFR expression.

(A–C) U937 cell lines expressing GFP or GFP.VentX under the control of tetracycline-inducible promoter were treated with 1.0 μg/ml DOX for 72 hours. (A) Western blot analysis of M-CSFR protein levels from U937 cell lysates. Tubulin was used as a loading control. (B) Surface expression of M-CSFR was determined by FACS analysis. Filled gray histogram represents isotype control; solid line histogram represents cells expressing GFP; dotted line histogram represents cells expressing GFP.VentX. (C) M-CSFR mRNA levels were determined by real-time PCR. Data represent mean + SD of triplicates from 1 representative experiment. (D) VentX transactivation of M-CSFR promoter. pcDNA-VentX or pcDNA-control was cotransfected with wild-type or mutant M-CSFR promoter luciferase reporter constructs into U937 cells. The effect of VentX on M-CSFR promoter transactivation was determined by luciferase activity. Data are mean + SD of triplicates from 1 representative experiment. (E) ChIP analysis of the interaction between VentX and the M-CSFR promoter, showing the association of VentX with the M-CSFR promoter region but not with the Cμ region in U937 cells. (F) Gel shift analysis showing the binding of VentX to the wild-type M-CSFR promoter probe, but reduced binding to the mutant M-CSFR promoter (Mut) probe. *P < 0.05, **P < 0.01.