Figure 5

DNMT1 mediates VLDL-induced de novo DNA methylation. A, RT-PCR analysis of DNMT expression in siRNA-treated cells stimulated with two preparations of VLDL at 58 μg protein/ml. B, effects of siRNA-mediated knockdown of DNMT1 or DNMT2 on total 5 mdC content. Solid squares, control; open squares, DNMT2-specific siRNA; solid diamonds, DNMT1-specific siRNA ID no. 110915; open diamonds, DNMT1-specific siRNA ID no. 110917 (both from Ambion). Asterisks indicate statistically significant comparisons between siRNA-treated and control cells. For simplicity, when cells treated with either DNMT1-specific siRNA show different significance levels, only the lower one is shown (*, P < 0.05; ***, P < 0.001). C, effects of siRNA treatment on the methylation status of IL6 and IL1B, as assessed by COBRA. DNAs from the two DNMT1-specific siRNAs were pooled for this analysis. Statistical significance is indicated as in B. Bars, standard deviations. D, VLR alters baseline phosphorylation of PKC zeta and Raf-1. Fields of Kinexus Kinetworks™ Phospho Site Screen KPSS-4.1 containing PKC zeta T410 and Raf-1 S259 in control and VLR-stimulated cells. Raf-1 migrates as multiple bands reflecting multiple phosphorylation states [55]. Positions of relevant MW standard are shown on the left (kDa).