Fig. 3.

Camk2g promoter is methylated in vascular smooth muscle (VSM) synthetic phenotype (VSM_Syn) cells. A: diagram of methylation DNA immunoprecipitation procedure. Genomic DNA (gDNA) is extracted from tissues or cultured cells and fragmented to approximate 500 bp by sonication. A specific methylated cytosine antibody is used to pull down methylated gDNA fragments and the presence and enrichment of specific genes are examined by quantitative PCR (qPCR). B: gDNA extracted from cultured VSM_Syn cells was fragmented and immunoprecipitated. The presence of Camk2 isoform promoters in the methylated gDNA fraction was tested by PCR amplification of ⁻621 to ⁻472 in the Camk2g promoter and ⁻577 to ⁻442 in the Camk2d promoter. C: enrichment of Camk2g and myosin heavy chain (Myh11) in the methylated gDNA fractions was compared between aorta tissue with differentiated VSM (VSM_Diff) and cultured VSM cells (VSM_Syn). D: VSM_Syn cells were treated with 5-Aza-2'-deoxycytidine (5-Aza) (10 μM) for 5 days and the enrichment of methylated Camk2g and Myh11 compared with untreated and treated cells. Values are shown as means ± SE, n = 3–4, and analyzed by 2-way ANOVA with Tukey post hoc test. *P < 0.05 and **P < 0.01. Ca²⁺/calmodulin-dependent protein kinase IIγ, CaMKIIγ.