Figure 5

Mel-18 is a positive regulator of cytokine genes in D5 and D10 cells. (A) ChIP experiment assessing the binding of Mel-18 at the Ifng (left) and Il4 (right) promoters in D5 and D10 cells. The cells were stimulated with antigen-presenting cells (APCs) with the appropriate peptides, cultured for 2-3 weeks, and then left unstimulated or were re-stimulated (P+I) for the indicated time points. In the panel of the Ifng promoter the result of 1 hr stimulation of D5 cells was set as 1, and in the Il4 promoter panel the result of 1 hr stimulation in D10 was set as 1. (B) Quantitative RT-PCR for the indicated mRNAs following Mel-18 knockdown in D5 (left) and D10 (right) cells. The cells were re-stimulated with APCs with the appropriate peptides, transduced with lentiviral shRNAs, cultured in the presence of puromycin for 2-3 weeks, and then re-stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours. The results are presented relative to the control, defined as 1. Differences between knockdown and control with p values ≤ 0.05 are indicated with an asterisk (C) Intracellular staining of Mel-18 in the indicated transduced D5 and D10 cells. (D) Quantitative RT-PCR for Mel-18 in resting and 2-hour-restimulated cells as indicated. The expression level in resting Th1 cells was set as 1. The results in Figure 5 are the mean of three to six independent experiments +S.D., except panel 5C, which is a representative experiment.