Figure 4. HXKII gene expression is dependent upon JNK/JunD signaling.

(A) HXKII gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in Methods. Cells were cultured with or without IL-7, or after an IL-7 pulse for 2 hours (IL-7 Re-Addition), in presence of a vehicle control, 20 µM JNK inhibitor, or 20 µM p38 inhibitor. RQ (Fold change) = 2^−ΔΔCt. (***) indicates P<0.001. (B) HXKII gene expression in D1 cells cultured with or without IL-7 and the non-targeting control (NT) or JunD siRNA, as described in Methods, was measured as above. (*) indicates P = 0.0254. Efficacy of JunD siRNA upon JunD mRNA levels (right panel) was established through measuring total JunD gene expression by quantitative PCR. (*) indicates P = 0.0336. RQ (Fold change) = 2^−ΔΔCt. (C) Chromatin Immunoprecipitation (ChIP) was performed using nuclear lysates from D1 cells cultured with or without IL-7 for 18 hours. Results from the quantitative PCR, reported as cycle threshold (Ct) values, are shown in the table. The PCR-amplified 150 bp region of AP-1 promoter DNA from the HXKII gene was visualized by ethidium bromide staining in a non-denaturing agarose gel. Input DNA is shown as equivalent starting materials. Results (A, B and C) are representative of three experiments performed in triplicate (values in graphs are mean ± SD).