Figure 1.
Modulation of DEFB1 gene expression and histone H3 acetylation by the HDAC inhibitor TSA.
The human lung epithelial cell lines A549 (A) and NCI-H727 (B–C) were treated with the HDAC inhibitor trichostatin A for 24 h. (A–B) mRNA expression of DEFB1 was analyzed using quantitative Real-Time-PCR. The measured levels of mRNA were normalized to PBG-D mRNA levels. (C) ChIP analysis was performed using anti-acetyl-Histone H3 antibody. Isolated DNA was quantified using quantitative RT-PCR. Bars represent means with SD of three independent experiments. LINE: long interspersed nuclear element.
Figure 2.
Modulation of DEFB1 gene expression by HDAC inhibitors.
The human lung epithelial cell line A549 was treated with HDAC inhibitors and IL-1β for 18 to 48 h. mRNA expression of DEFB1 (A) and IL8 (B) was analyzed using quantitative Real-Time-PCR. The measured levels of mRNA were normalized to TBP mRNA levels. DEFB1/IL-8 mRNA expression was normalized to control treatment. Bars represent means with SD of three independent experiments (*: p<0.05; **: p<0.01; ***: p<0.001).
Table 1.
Specificity of HDAC inhibitors.
Figure 3.
The HDAC inhibitor MS-275 increases H3 acetylation and H3K4 trimethylation at the DEFB1 promoter. (A) DEFB1 gene with promoter region including analysis sites I-IV. I: −186 – −250, II: −347 – −406, III: −1002 – −1061, IV: −2436 – −2499.
(B–C) A549 cells were treated with HDAC inhibitor MS-275 for 36 h. ChIP analysis was performed using anti-acetyl-Histone H3 (B) and anti-trimethyl-H3K4 (C) antibodies. Isolated DNA was quantified using quantitative RT-PCR. Bars represent means with SEM of three independent experiments. LINE: long interspersed nuclear element.
Figure 4.
Knockdown of HDAC1 increases DEFB1 gene expression.
A549 cells were transfected with HDAC and control siRNAs for 48 to 96 h. (A) Cells were lysed 48 h after transfection and Western Blot analysis was performed with an anti-HDAC1 antibody. Data are representative of three independent experiments. (B–F) mRNA expression of Cyclophilin B (B), HDAC1 (C), HDAC2 (D), HDAC3 (E) and DEFB1 (F) was analyzed using quantitative Real-Time-PCR. The measured levels of mRNA were normalized to TBP mRNA levels. Bars represent means with SEM of three independent experiments (*: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001).