The Jak/Stat3 pathway promotes the expression of IL-17F in malignant T cells. (A) Malignant T cells (PB2B) were incubated with Jak inhibitors (Jak3 inhibitor II/WHI-P154, 40 µM; Jak inhibitor I/P6, 1 µM), an inhibitor against Stat3 (Sta-21, 40 µM), an inhibitor against the EGF receptor (Ag1478, 200 ng mL^–1) or vehicle (dimethylsulfoxide) and the expression of phosphorylated Stat3 (pYStat3), total Stat3, and Erk 1/2 analyzed by western blotting. (B) Malignant T cells (PB2B) were cultured with inhibitors as described above for 4 hours. Subsequently, the cells were harvested and the relative levels of IL-17F and GAPDH mRNA determined by QPCR. In each sample, the level of IL-17F mRNA was normalized to that of GAPDH mRNA and depicted as the fold change compared with cells cultured with vehicle. (C) Malignant T cells (PB2B) were cultured for 24 hours with inhibitors as described above and the concentration of IL-17F in the cell culture supernatants determined by enzyme-linked immunosorbent assay. The expression of IL-17F is shown as the percent expression relative to cells treated with vehicle. (D) Jurkat T cells were cotransfected with a luciferase reporter plasmid containing the proximal human IL-17F promoter and a Renilla luciferase plasmid as well as either an empty pcDNA3.1 plasmid or a pcDNA3.1 plasmid encoding a constitutive active form of Stat3 (Stat3c). At 24 hours after transfection, the cells were lysed and the luciferase activities determined. The coexpressed Renilla luciferase activity was used for normalization of transfection efficiency. Bars represent mean + SEM.