Figure 5.

Inhibition of ACLY by HCA attenuated IL-1β–induced augmentation of acetylation H3K9 and H3K27 both globally and specifically in the iNOS, MMP3, and MMP13 promoters. Cultured primary human knee chondrocytes were treated with IL-1β (2 ng/ml) with or without HCA (20 mm) for 5 h. Nucleocytosolic acetyl-CoA was measured (A). Phosphorylation (Ser-455) and expression of ACLY and acetylation of H3K9 and H3K27 (B), and acetylation of p65 NF-κB (Lys-310) and phosphorylation of p65 NF-κB (Ser-536) (D) were examined by Western blotting, with β-actin and histone H3 as loading controls. Semi-quantitative densitometry analysis of each Western blotting was performed over 3 individual experiments with 3 different donors (B and D). ChIP-qPCR assessed Ac-H3K9 and Ac-H3K27 occupancy of iNOS, MMP3, and MMP13 promoters, and were presented as percentage of input of sonicated genomic DNA (C). Data in C represent the mean ± S.D. of 3 individual experiments with 3 different donors. Two-way ANOVA followed by Bonferroni's post hoc test (A and C) and Student's t test (B and D) were used for statistical data analysis. p values represent comparisons of the mean ± S.D.