Figure 7

Iloprost suppressed poly I:C-induced TNF-α expression in mDCs via histone H3K4 trimethylation in the TNFA gene promoter region. Isolated mDCs were pretreated with iloprost for 2 h and then stimulated with poly I:C for 1 h and were used for ChIP assay using anti-H3K4 antibodies. The cells were also used for cytosolic and nuclear protein analysis by Western blotting using anti-MLL and anti-WDR5 antibodies. The nuclear and cytosolic fractionation technique was verified using anti-α-tubulin and anti-histone H3 antibodies. (A) Iloprost downregulated poly I:C-induced H3K4 trimethylation in the TNFA gene promoter region. (B) Iloprost suppressed poly I:C-induced translocation of cytosolic H3K4-specific methyltransferases MLL and WDR5 proteins into nucleus. The nuclear fraction is lack of α-tubulin, and the cytosolic fraction is lack of H3. To investigate whether MAPK-p38 signaling is responsible for the translocation of MLL and WDR5 from cytoplasm to nucleus, mDCs were pretreated with MAPK-p38 inhibitor SB203580 for 2 h and then stimulated with poly I:C for 1 h. (C) SB203580 suppressed poly I:C-induced translocation of MLL and WDR5 from cytoplasm to nucleus. (D) Densitometry analysis of the Western blot data shown in (B). Results represented are the mean ± SD of three independent experiments using mDCs from three subjects for the ChIP assay and densitometry analysis. For Western blotting analyses, one experiment representative of three is shown. *P ≤ 0.05 compared with poly I:C-treated cells (A and D). OD, optical density. A: □, Control; ■, poly I:C; ■, iloprost + poly I:C.