IL-6-activated Stat3 binds to the ROR1 promoter and activates transcription in MM1 cells.
A. ROR1 promoter biotinylated DNA probe binds to nuclear protein of unstimulated or IL-6-stimulated MM1 cells. The addition of an unlabeled (cold) probe attenuated the binding of the hot (biotin-labeled) probe. Similarly, anti-Stat3 (total Stat3) and anti-phosphotyrosine-Stat3 antibodies attenuated the binding of the hot probe, suggesting that nuclear Stat3 protein bound the labeled ROR1 probe. B. ChIP demonstrates that anti-(total) Stat3 antibodies immunoprecipitated ROR1, STAT3, and the Stat3-regulated genes c-Myc and p21. These genes were detected in MM1 cell nuclear extracts (Input) as well as in nuclear protein immunoprecipitated with anti-Stat3 antibodies, particularly in IL-6-stimulated MM1 cells, suggesting that Stat3, known to bind Stat3-regulated gene promoters, also binds the ROR1 promoter. C. qRT-PCR demonstrates that STAT3-siRNA downregulates the mRNA levels of ROR1, STAT3, and the Stat3-regulated genes c-Myc, Cyclin D1, and p21. S18 mRNA (control) was not affected. D. Western blot analysis shows that STAT3-siRNA, but not scrambled-siRNA or GAPDH, downregulates the protein levels of Stat3 and Ror1.