FIGURE 4.

Mechanism of BAMBI repression by HDAC1. A, the knockdown efficiencies of siRNA HDAC1 were estimated by Western blot analysis. LX2 cells were transected with siRNA HDAC1 or a control for 72 h, followed by treatment with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 4 h. B, LX2 cells were transfected with siRNA HDAC1 for 72 h. Then, hBAMBI mRNA was measured by qPCR. 18 S was used as an internal control for normalization. Un, untreated. Data represent mean ± S.D. *, p < 0.05. C, effects of LPS and TNF-α on the recruitment of HDAC1 to the κB binding site in the hBAMBI promoter were assessed. LX2 (left panel) and hHSCs (right panel) were treated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 2 h. A ChIP analysis using anti-HDAC1 or control Ab (normal rabbit polyclonal IgG) was performed. BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. D, effects of TSA on the recruitment of HDAC1 to the κB binding site in the hBAMBI promoter were analyzed. LX2 cells were pretreated with TSA (300 nm/ml) for 8 h and then treated with LPS or TNF-α for 2 h. A ChIP analysis using anti-HDAC1 or control Ab (normal rabbit polyclonal IgG) was employed. BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. E, effects of NF-κB inhibition on the binding of HDAC1 to the κB binding site in the hBAMBI promoter. LX2 cells were treated with control Ad-GFP or Ad-IκBsr overnight and then treated with LPS or TNF-α for 2 h. A ChIP analysis on the BAMBI promoter using anti-HDAC1 or control Ab (normal rabbit polyclonal IgG) was performed. F, ChIP analysis for the assessment of the effects of TSA on the binding of p50 to the κB binding site in the hBAMBI promoter. G, direct interaction of p50 with HDAC1 after LPS or TNF-α stimulation was assessed. LX2 cells were treated with control Ad-GFP or Ad-IκBsr overnight and then treated with LPS and TNF-α for 2 h. After immunoprecipitation (IP) with anti-p50 antibody, Western blotting (WB) for HDAC1 was performed.