EPA reduces Mmp9 expression in macrophages.

A. Gelatin zymography of aortic tissues one week after CaCl₂ 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, n = 6–7 in each group. B. Gating strategy for the flow cytometric analysis of AAA macrophages. Macrophages were identified as Ly-6C^lowCD11b⁺F4/80⁺Ly-6G⁻ cells (full gating strategy shown in Figure S2 in File S1). C. 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. D. The mRNA levels of Mmp9 in sorted aortic macrophages. Expression levels were first normalized to 18s rRNA levels and then further normalized to the level of control diet group. n = 5 in each group. E. 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 18s rRNA levels and then presented as relative expression compared to baseline vehicle sample. n = 3 per condition. *P<0.05 compared to control diet group in A and D or respective vehicle controls in E.