Influence of intron 1 CpG island B demethylation on RNAPII and p53 binding to H-Ras gene.

<p>(A) Schematic representation of H-Ras exon 1, intron 1, and exon 2. (B) ChIP was performed in control (<i>white bars</i>), OA (<i>gray bars</i>), and EPA treated (<i>black bars</i>) U937 cells, using RNAPII Ab. qRT-PCR was performed using specific primers for exon 1, intron 1 region C, CpG island B, intron 1 region D, and exon 2. The results shown are the mean ± SD of three independent experiments. (C) ChIP was performed as in (B) using a p53 Ab. The region within CpG island B containing the p53 element was amplified by qRT-PCR. The results shown are the mean ± SD of three independent experiments. (D) PCR of DNA from p53 Ab immunoprecipitated complex. Input, fragmented DNA before immunoprecipitation, negative controls rIgG. One representative out of three experiments is shown.</p

Effect of EPA on Ras isoforms expression.

<p>mRNA content was evaluated for H-Ras, N-Ras, and K-Ras after 1-, 3-, and 24-h treatment with 100 µM fatty acids, using qRT-PCR. <i>White bars</i>, control U937; <i>gray bars</i>, OA; <i>black bars</i>, EPA. Data are presented as relative expression by calculating 2^−ΔΔCt normalized to untreated U937 cells. The means ± S.D. of three separate experiments are shown (*, p<0.01).</p

Effect of fatty acids on Ras, ERK1/2, and phospho-C/EBPβ protein levels.

<p>(A) U937 cells were treated with 100 µM fatty acids (OA, oleic; LA, linoleic; LNA, α-linolenic; AA, arachidonic; EPA, eicosapentaenoic; DHA, docosahexaenoic) for 24 hours. Total cell lysates or isolated non raft membrane fractions (50 µg protein) were subjected to Western blotting with the indicated antibodies. Pan-Ras Ab was used to detected all Ras isoforms. For each protein, one representative out of three experiments is reported. (B) Quantitative analysis. The chart shows normalized Western blot band densities, presented as fold induction with respect to U937 control cells. Images of independent blots were acquired using the Versadoc Imaging System and signals were quantified using Quantity One Software. Data are the means ± S.D. of three independent experiments. (*, p<0.05)</p

EPA increases H-Ras exon 2 transcription and demethylates intron 1 CpG island B.

<p>(A) mRNA content was evaluated for H-Ras exon 1 and exon 2 after 1-,3-, and 24-h treatment with 100 µM fatty acids using qRT-PCR. <i>White bars</i>, control U937 cells; <i>gray bars</i>, OA; <i>black bars</i>, EPA. Data are presented as relative expression by calculating 2^−ΔΔCt normalized to untreated U937 cells. The means ± S.D. of three separate experiments are shown (*, p<0.01). (B) H-Ras intron 1 CpG island B sequence. The underlined sequences indicate the forward and reverse primers utilized for cloning and sequencing after bisulfite reaction. The cloned fragment (242 bp) contains 26 CpGs (in bold). The nucleotide sequences outside the CpG island B are in italics. (C) Sequencing of individual clones generated by PCR after bisulfite reaction. Black and white square represent methylated and unmethylated CpGs, respectively.</p

Fatty acid composition of total lipids from non raft membranes fraction.

<p>Fatty acid composition of total lipids from non raft membranes fraction.</p

Fatty acid composition of total lipids from raft membranes fraction.

<p>Fatty acid composition of total lipids from raft membranes fraction.</p