Figure 2

Histone H3K9 trimethylation is reduced in stimulated naive BMMCs and inhibition of histone demethylation leads to reduced cytokine production. BMMCs were pretreated with LPS (300 ng/ml) for 24 h to induce ET. To analyse histone modifications in naive and tolerant cells, the cells were stimulated with LPS (1 µg/ml) for 30 min and 60 min and ChIP analysis was performed. Fold change in site occupancy of H3K4me3 (a) and H3K9me3 (b) at the Il6 and Tnf gene was measured. Data show mean ± SD from n = 3 independent experiments. Two-sided ANOVA with Tukey HSD post-hoc test and one-sample t-Test were performed to calculate the p-values. Multiple testing p-values were corrected by FDR. Localisation of PCR products within the Il6 gene and the Tnf gene are depicted. Furthermore, cells were incubated with the LSD1 inhibitors Pargyline (1000 µM) or TCP (250 µM) for 2 h and stimulated with LPS (2 µg/ml) for 4 h to analyse protein production of IL-6 and TNF-α (c). Data show mean ± SD from n = 3 independent experiments with three biological samples. Student’s t-Test was performed to calculate the p-values. *p < 0.05, **p < 0.01, ***p < 0.001.