FIGURE 10.

The effects of dexamethasone on the recruitment of GR, ACTN4, and p65 to NF-κB targeted promoters. A, diagrams of mapped NREs or putative GRE and PCR primer sets. Dark gray squares indicate p65 or GR binding sites on NF-κB-inducible genes. Light gray squares indicate NF-κB sites described in our previous paper (27). B–D, HPCs were treated with vehicle or dexamethasone (Dex) for 4 h, followed by ChIP assays with anti-ACTN4, anti-GR, and anti-p65 antibodies. Immunoprecipitated (IP) chromatin was analyzed by qPCR using the primers (A, small arrows) flanking the putative p65-binding sites in the promoters of IL-1β, IL-6, and IL-8. E, control and ACTN4 knockdown HPCs were treated with vehicle or dexamethasone for 8 h prior to harvest. Total RNA was extracted, and quantitative RT-PCR analysis was conducted using gene-specific primers. F–H, HPCs stably expressing shCtrl or shACTN4 were treated with vehicle or dexamethasone for 4 h prior to harvest. The relative recruitment of ACTN4, GR, and p65 to the promoters of IL-1β, IL-6, and IL-8 was determined by normalizing the PCR products from shACTN4 cells to that of shCtrl cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant.