FIG. 5.

(A) The histone methylation inhibitor MTA markedly decreased the ability of the cells to produce TNF-α, as detected by ELISA. The results are expressed as a percentage of the TNF-α produced by each stimulus in the absence of the MTA inhibitor. “IC” represents rabbit anti-bovine serum albumin immune complexes at 1 μg/ml. MTA diminishes the response to all stimuli. (B) The methylation inhibitor MTA reduced the ability of the cells to produce TNF-α by flow cytometry. Only 0.6% of the cells were positive by flow cytometry when unstimulated. After treatment with 1 μg/ml of LPS for 6 h, 47.6% of the cells were positive and had a mean fluorescent intensity of 56.3 U (black line). MTA pretreatment followed by LPS stimulation led to 19.5% of the cells being positive, with a mean fluorescent intensity of 39.9 U (gray fill). The black fill represents unstimulated, untreated cells. (C) ELISA study of siRNA-transfected THP-1 cells. Cells were stimulated for 6 h with 100 ng/ml of LPS 48 h after transfections, and supernatants were tested for TNF-α. siRNAs directed at Ash2L and RbBP5 have been shown to compromise H3 lysine 4 dimethylation and trimethylation. (D) The two MLL knockdown siRNAs and siRNA to GAPDH were transfected in to THP-1 cells in parallel cultures. The data are expressed as the signal in the Ash2L- and RbBP5-transfected cells normalized to GAPDH. H3 lysine 4 dimethylation was increased in the MLL knockdowns in the 3′ region of LTα and clearly decreased in the TNF-α third intron enhancer region.