Figure 5.

Roles of FoxO3a in TNF-α-induced CYR61 expression and effects of simvastatin on FoxO3a. (a) After transfecting GO orbital fibroblasts with FoxO3a-expressing plasmid, overexpression of FoxO3a in transfected orbital fibroblasts was confirmed by western blot analysis. (b) Original orbital fibroblasts and transfected orbital fibroblasts with overexpression of FoxO3a were exposed to 20 ng/ml TNF-α for 24 h with or without pretreatment with 10 μM simvastatin for 3 h. CYR61 protein production was assessed by western blot analysis. (c) GO orbital fibroblasts were incubated with 20 ng/ml TNF-α for the indicated time points. The p-FoxO3a protein in total cell lysates was examined by western blot analysis. (d, e) GO orbital fibroblasts were exposed to 20 ng/ml TNF-α for 15 min with or without pretreatment with 10 μM simvastatin for 1 h. The levels of FoxO3a in the nuclear fractions (d) and p-FoxO3a in the cytoplasmic fractions (e) were measured by western blot analysis. (f) GO orbital fibroblasts treated with 20 ng/ml TNF-α for 15 min with or without pretreatment with 10 μM simvastatin were subjected to chromatin immunoprecipitation (IP) with anti-FoxO3a antibodies. The immunoprecipitated DNA were evaluated by real-time PCR using primers for the region of the CYR61 promoter flanking the FoxO3a binding site. Data are presented as mean ± SD of at least three independent experiments. ^∗p < 0.05.