Figure 5

DNMT1 regulates the OS-induced expressions of CTGF and cyclin A via directly targeting their promoters. (a) ECs were infected with adenovirus expressing shDNMT1 or treated with DNMT inhibitor 5-Aza-2′-deoxycytidine (5-Aza, exposed to PS or OS for 6 hours, and expressions of DNMT1, cyclin A, and CTGF were assessed by Western blot. (b) ECs were exposed to PS or OS for 6 hours. After that, DNMT1 binding at CTGF and cyclin A promoter regions was analyzed by chromatin immunoprecipitation (ChIP) assay. (c) CpG island search in the promoter regions of cyclin A and CTGF. The shade shows regions with GC percentage greater than 50%. The locations of the methylation-specific PCR (MSP) primers are illustrated. (d) ECs were exposed to PS or OS for 6 hours. After that, the methylation levels of the CTGF and cyclin A promoter were measured by MSP. M: methylated; U: unmethylated. (e) ECs were infected with control virus or ad-hshDNMT1, or transfected with control plasmid or plasmid overexpressing DNMT1. Expressions of DNMT1, cyclin A, and CTGF were analyzed by Western blot. (f) Immunofluorescent-staining of cyclin A and CTGF in unligated and ligated carotid arteries from C57B/L6 wild-type (WT) mice at one week after operation. The ligated carotid arteries were subjected to a locally intraluminal incubation with ad-mshDNMT1 or control virus. (g) Immunofluorescent-staining of cyclin A and CTGF in unligated and ligated carotid arteries from ApoE^−/− mice at four weeks after operation. Images are representative of 3 independent experiments or 8–10 mice with similar results. *P < 0.05 vs. PS.