Figure 4.

STAT3 activates the GM-CSFRα promoter in chronic lymphocytic leukemia (CLL) cells. (A) STAT3 binds to the promoter of GM-CSFRα and other STAT3 regulated genes. ChIP demonstrates that anti-STAT3 antibodies immunoprecipitated GM-CSFRα (left panel) and the STAT3-regulated genes STAT3, c-Myc, WAF1/p21, ROR1, and VEGF (right panel) in CLL cells. These genes were detected in CLL nuclear extracts (Input) as well as in nuclear protein immunoprecipitated with anti-STAT3 antibodies, suggesting that STAT3 binds to known STAT3-regulated gene promoters and to the GM-CSFRα promoter. As shown in the left panel, binding of STAT3 to the GM-CSFRα promoter is detected by primers 1, 3, and 5, but not 2 and 4. (B) EMSA results, using biotin-labeled and unlabeled fragments 1, 3, and 5 and their corresponding mutant probes (Supplementary Table 2), show that CLL nuclear protein from 2 different patients binds to the biotinylated DNA fragments 1, 3, and 5 and that the addition of the corresponding cold (unlabeled) DNA fragments or anti-STAT3 antibodies (but not IgG) attenuates this binding. The binding of the labeled mutant DNA probes was diminished or significantly reduced compared with that of the unlabeled mutant DNA probes. (C) qRT-PCR demonstrates that lentiviral STAT3-shRNA downregulates mRNA levels of GM-CSFRα and the STAT3-regulated genes STAT3, Bcl-2, Bcl-X_L, cyclin D1, c-Myc, and WAF1/p21. Expression of S18 mRNA (control) was not affected. (D) Western immunoblotting shows that STAT3-shRNA reduced GM-CSFRα protein levels. The empty vector reduced STAT3 protein levels by 10% and GM-CSFRα protein levels by 40%, whereas STAT3-shRNA downregulated STAT3 by 80% and GM-CSFRα protein levels by 90%.