Figure 1

Knockdown of Mel-18 downregulates the expression of cytokine genes in primary Th1 and Th2 cells. (A) Quantitative RT-PCR for Mel-18 in naïve cells, differentiating Th1 and Th2 cells (days 2, 6, and 8), and 8^th-day differentiated Th1 and Th2 cells after 1 or 2 hours of re-stimulation with P+I. The expression level in Th1 cells on day 2 was set as 1. (B,C) Quantitative RT-PCR for the indicated mRNAs following Mel-18 knockdown by Mel-18-directed shRNAs M1, M2 or control non-silencing scrambled shRNA during Th1 (B) and Th2 (C) differentiation. The results are presented relative to the control, defined as 1. The cells were transduced on day 0, and after 3 days of puromycin selection, on the 5^th day, they were re-stimulated with P+I for 2 hours. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. (D) Intracellular staining of Mel-18 in the indicated transduced Th1 and Th2 cells. (E) ELISA for the levels of IFNγ and IL-4 in the supernatants of 4-hour-stimulated (αCD3 and αCD28) Th1 and Th2 cells, respectively, after Mel-18 knockdown. The results with control shRNA were set as 1. (F) Western blot assessing the indicated proteins on the 5^th day of Th1 and Th2 cell transduced with control shRNA [C] or shRNA directed to Mel-18 [M1]. c-jun is used as loading control. (G) Quantitative RT-PCR for the indicated mRNAs after Mel-18 knockdown in Th1 (left) and Th2 (right) cells as described in Figure 1B, except the cells were re-stimulated with αCD3 and αCD28 antibodies. Differences between knockdown and control with p values ≤ 0.05 are indicated with an asterisk. The results in Figure 1 are the mean of two to five independent experiments +S.D., except in panels 1D and 1F, which are representative experiments.