FIGURE 4.

Inhibitory effect of PUFA on Srebf1c gene transcription is mediated through SREBP-1 itself. A–H, knockdown of hepatic SREBP-1 (A–D) or SREBP-2 (E–H) by adenoviral expression of shRNA. 250- or 90-bp Srebf1c-Luc adenovirus (Ad-250bp- Srebf1c-Luc or Ad-90bp- Srebf1c-Luc; 6.0 × 10⁶ pfu/body) plus adenovirus expressing SREBP-1-specific, SREBP-2-specific, or LacZ-specific shRNA (Ad-SREBP1i, Ad-SREBP2i, or Ad-LacZi; 2.5 × 10⁸ pfu/body) were intravenously injected into ICR male mice. After 4 days, the mice (n = 3–6 for each group) were treated orally with 7.5 g/kg EPA or water (CTL) once a day for 4 days. On days 0 and 4 after the first treatment of EPA, luciferin was injected intraperitoneally, and the luminescence from liver was captured with IVIS. A, B, E, and F, quantification of luciferase activity with LivingImage software. Fold changes of luciferase activity on day 4 versus day 0 are shown. C and G, Northern blot analysis of SREBP-1 and -2 in the liver. Total RNA (7.5 μg) from livers pooled equally from mice for each group was subjected to Northern blotting to determine SREBP-1, -2, and 36B4 (used as a loading control) mRNA levels. D and H, immunoblot (IB) analysis of mature and precursor SREBP-1 and -2 proteins in the liver. Aliquots of nuclear extracts (10 μg) and total proteins (50 μg) from livers pooled equally from four male mice of each group were subjected to immunoblot analysis. The primary antibodies used were polyclonal anti-mouse SREBP-1 and polyclonal anti-mouse SREBP-2. These data are representative of at least two independent experiments (n = 3–6 mice/group). Results are means ± S.E. *, p < 0.05, and **, p < 0.01, respectively.