HDAC inhibitors increase Mi-2β expression and recruitment to TSA-sensitive promoter. (A-I) BMDMs were preincubated for 1 hour with (+) or without (−) TSA (100nM unless specified) and exposed to LPS (100 ng/mL) or Pam3CSK4 (100 ng/mL) for the indicated time or 1 hour (E). NF-κB DNA binding activity and NF-κB p65, c-jun, phosphorylated (P-), and total ERK1/2 and p38 and P-STAT1 expression were analyzed by electrophoretic mobility shift assay (A) and Western blot (B-D) using nuclear (nuc) and cytosolic (cyto) extracts. The retarded complex detected by electrophoretic mobility shift assay was dose-dependently inhibited by cold wild-type but not mutant NF-κB oligonucleotide, and supershifted using anti-p65 antibody (data not shown). Acetylation of histone H4 (E) and Mi-2β recruitment (I) to Tnf and Il6 promoters were analyzed by chromatin immunoprecipitation. Mi-2β and BRG1 mRNA and protein levels were quantified by real-time PCR (F) and Western blot (G) with densitometric analyses (H). Data are representative of 2 to 5 independent experiments. (J) RAW 264.7 macrophages transfected with control (Ctl), Mi-2β (Mi), or BRG1 (Br) siRNAs. After 3 days, cells were incubated for 4 hours with (+) or without (−) 10 ng/mL of LPS. Il6 and Tnf mRNA levels were analyzed by RT-PCR and results expressed as the ratio of cytokine to GAPDH mRNA levels. Data are representative of triplicate determinations from 1 experiment.
