Figure 4.
HDAC2 suppression by siRNA and its role in mediating the effect of budesonide on human lung fibroblasts and bronchial epithelial cells.
Notes: HFL-1 cells or HBECs were transfected with either a negative control siRNA or an siRNA targeting HDAC2 (HDAC2-siRNA). Cells were then treated with cytokines IL-1β plus TNF-α (1 ng/mL each) in the presence or absence of budesonide (10−7 M). Hatched bars: cells transfected with non-targeting control-siRNA; solid bars: cells transfected with siRNA specifically targeting HDAC2. (A) Suppression of HDAC2 by siRNA in HFL-1 cells. Density of HDAC2 was normalized by β-actin density for each experiment, and then data were expressed a mean ratio (± SEM) of HDAC2-siRNA density versus control-siRNA density in three separate experiments. Inset: Image of immunoblot from one representative experiment. Top band: HDAC2; bottom band: β-actin. **P < 0.01 compared to control-siRNA. (B and C) Effect of HDAC2 suppression by siRNA on budesonide (10−7 M) inhibition of IL-6 and IL-8 release (ELISA) by HFL-1 cells (data were normalized to the cell number; data are shown as the mean ± SEM of three separate experiments). (D, F and G) Effect of HDAC2 suppression by siRNA on budesonide (10−7 M) inhibition of MMP-1 and MMP-3 release (immunoblotting) by HFL-1 cells. Data presented are one representative of three separate experiments (D) or the mean ± SEM of three separate experiments. In order to make quantitative comparisons, the same volume of media from each condition was harvested and precipitated for loading. (E and H) Effect of HDAC2 suppression by siRNA on budesonide (5 × 10−7 M) inhibition of IL-6 release by HBECs. After siRNA transfection, cells were treated with cytokines and budesonide as indicated for 24 hours. Media were harvested and IL-6 was quantified by ELISA (data were normalized to the cell number). (E) Immunoblots of HDAC2 and β-actin, showing suppression of HDAC2 by HDAC2-siRNA but not by control-siRNA. (H) Vertical axis: IL-6 amount expressed as percent of cytokine in control-siRNA treated cells; horizontal axis: treatment. Data presented are the mean ± SEM of two different HBEC cell lines. *P < 0.05, **P < 0.01.
Abbreviations: Bud, budesonide; Cyto, cytokines; ELISA, enzyme linked immunosorbent assay; HBEC, human bronchial epithelial cells; HDAC, histone deacetylase; HFL, human lung fibroblast; IL, interleukin; MMP, matrix metalloproteinase; SEM, standard error of the mean; si, small interfering; TNF, tumor necrosis factor; TSA, trichostatin A.