EPA reduces <i>Mmp9</i> expression in macrophages.

<p><b>A</b>. Gelatin zymography of aortic tissues one week after CaCl<sub>2</sub> treatment together with quantitative analysis, showing reduced MMP9 activity in samples from the EPA diet group. Equal amounts of protein (20 µg) were loaded per aortic sample. For quantitation, <i>n</i> = 6–7 in each group. <b>B</b>. Gating strategy for the flow cytometric analysis of AAA macrophages. Macrophages were identified as Ly-6C<sup>low</sup>CD11b<sup>+</sup>F4/80<sup>+</sup>Ly-6G<sup>−</sup> cells (full gating strategy shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096286#pone.0096286.s001" target="_blank">Figure S2 in File S1</a>). <b>C</b>. The number of aortic macrophages per aortic sample. No statistically significant difference in the number of aortic macrophages between control diet and EPA diet groups was detected. <b>D</b>. The mRNA levels of <i>Mmp9</i> in sorted aortic macrophages. Expression levels were first normalized to <i>18s</i> rRNA levels and then further normalized to the level of control diet group. <i>n</i> = 5 in each group. <b>E</b>. RAW264.7 macrophages were cultured with either vehicle (10% BSA) or EPA (50 µmol/L) for 48 hours. The cells were then stimulated with recombinant mouse TNF-α (20 ng/mL) for a further 6 hours and harvested for analysis by RT-PCR. Expression levels were first normalized to <i>18s</i> rRNA levels and then presented as relative expression compared to baseline vehicle sample. <i>n</i> = 3 per condition. *<i>P</i><0.05 compared to control diet group in <b>A</b> and <b>D</b> or respective vehicle controls in <b>E</b>.</p>