TAK1 gene silencing results in the diminution of Th1 cells in vitro. Bone marrow–derived macrophages grown in GM-CSF (GM-BMMs) were generated as described in the Methods. GM-BMMs were first transiently transfected with siRNAs against TAK1 (siTAK1) or with a nontargeting control siRNA (siCT). After lipofection, the cells were stimulated with TNF (20 ng/mL) for 24 hours and then cultured with CD4⁺ T cells in the presence of anti-CD3 (1 μg/mL) for 5 days. (A) After lipofection, GM-BMMs stimulated with TNF were harvested and the expression of TAK1 was determined by qPCR. (B-C) IL-12 and IL-23 secretion in GM-BMM supernatants were measured by ELISA. (D) Flow cytometric analysis of Th1 and Th17 cells in CD4⁺ T cells cocultured with GM-BMM. Cells were stained for surface CD4 and intracellular IL-17A and IFN-γ. Representative dot plots were gated on CD4⁺ T cells. (E) Histograms indicate mean percentages of IL-17A and IFN-γ–producing CD4⁺ T cells. (F) IFN-γ and IL-17A production in the coculture of CD4⁺ T cells and GM-BMMs were measured by ELISA. Results are representative of 2 independent experiments performed in triplicate. *P < .05.