DNA methylation silences murine and human SPARC promoter activity. The mouse (a) and human promoter regions (b) were cloned into a pCPGL-basic plasmid (the CpG positions are indicated as balloons). There were no CpGs in the vector. The plasmids were either methylated or mock methylated in vitro and then transfected into HEK293 cells in the sense or antisense (1st column) configuration for 48 h (c, d). Relative luciferase activity (percentage) in the extracts is shown as average per group (triplicate transfection). The decrease in expression in the methylated (3rd column) vs. unmethylated (2nd column) treatment groups indicates gene silencing. One-way ANOVA followed by Bonferroni's test. ** = p < 0.01 Error bars = S.E.M.