Figure 4

Mild folate depletion induces epigenetic damage in prostate cells. (A) High-performance liquid chromatography analysis of adenosylmethionine/adenosylhomocysteine pools. Results are the average and standard deviation of three independent injections and are presented relative to cells grown in control medium (100%). * = P < 0.05, ** = P < 0.01, *** = P < 0.001. (B) Analysis of total levels of 5-methylcytosine by liquid chromatography-mass spectrometry. Results are the average and standard deviation of two independent injections. Values are relative to cells grown in control medium. (C) Sections of restriction landmark genomic scanning (RLGS) gels including the spot 4D20, which shows decreased intensity in both D100 and G100, and the spot 4G11, which shows decreased intensity in both G100 and H100. (D) Mass array quantitative methylation analysis confirmed the methylation status of CpG identified by RLGS and associated to known loci. Peripheral blood lymphocyte DNA was used as a 0% methylation control and an in vitro methylated aliquot of the same DNA as a 100% methylated control. These DNAs were mixed at appropriate ratios to generate 75%, 50% and 25% methylated controls. The figure shows 4D20 as an example. (E) Western Blot analysis of histone acid lysates run on sodium dodecyl sulphate-page gel revealed an increase of global H3K9 methylation (17 Kda) and H3K36 di-methylation (17 Kda) upon mild folate depletion.