Fig. 4.

Epigenetic regulation of Nox transcription by HDAC inhibition. (A) Agarose gel analysis of enzymatic shearing of chromatin from macrophages. L represents the 100 kb to 1000 kb DNA ladder, S1 and S2 represent sheared chromatin following enzymatic digestion for 5 and 10 min. (B) Macrophages treated with vehicle (DMSO) or scriptaid (3 μg/ml) were fixed with 1% formaldehyde for 10 mins and chromatin prepared by enzymatic shearing for 10 min. ChIP was performed on isolated sheared chromatin using a negative control IgG and ChIP grade antibodies to RNA pol II, H3K4 trimethylation (H3K4me3), H3K9 acetylation (H3K9ac) or p300. Real-time PCR was performed on DNA purified from each of the ChIP reactions using primers specific for the Nox2 promoter regions located −75 to −1 upstream of Nox2 transcriptional start site (TSS). (C) Agarose gel analysis of enzymatic shearing of chromatin from human lung fibroblasts. S1 and S2 represent sheared chromatin following enzymatic digestion for 10 min. (D-E) Human lung fibroblasts treated with vehicle (DMSO) or scriptaid (3 μg/ml) were fixed with 1% formaldehyde for 10 min and chromatin prepared by enzymatic shearing for 10 min. ChIP was performed on isolated sheared chromatin using a negative control IgG and ChIP grade antibodies to RNA pol II, H3K9 acetylation (H3K9ac) or p300. Real-time PCR was performed on DNA purified from each of the ChIP reactions using primers specific for the Nox4 and Nox5 promoter regions located +185 to +344 downstream, and −307 to −452 upstream, of the TSS, respectively. (F-G) Macrophages or human lung fibroblasts were treated with vehicle (DMSO) or scriptaid (3 μg/ml) for 24 h, and cell lysates were immunoblotted for p300 and GAPDH as a loading control. Data are expressed as mean ± S.E., *P<0.05 versus vehicle. (n=4–6). Results are representative of at least 3 separate experiments.