In vitro methylation of the NKAP and SPESP1 promoters decreased reporter gene activity. (A,B) The 1,500 bp promoter regions of NKAP (A) and SPESP1 (B) were inserted into the pCpGL basic vector. Promoter constructs were then methylated (gray and black bars) with HhaI or SssI or mock-methylated (white bar) before transfection into clonal β-cells for 48 h. After the 48 h transfection, the luciferase assay was run. The data were normalized with co-transfected renilla luciferase control vector and are the mean of five (NKAP) or nine (SPESP1) separate experiments of five replicates in each. Cells transfected with an empty pCpGL vector were used as background control for firefly luciferase results, and untransfected cells were used as a background for renilla luciferase results. Data were log-transformed and statistical tests calculated using ANOVA followed by paired t-tests with Bonferroni correction post hoc. Data are presented as mean ± standard error of the mean. *P <0.05 versus control; ¤P <0.05 versus SssI.