Figure 5.

Mevalonate overcomes the inhibitory effect of compactin on SREBP-1c mRNA (A) and protein (B) levels in McA-RH7777 cells. On day 0, cells were set up in medium A supplemented with 10% FCS at 7 × 10⁵ cells per 100-mm dish. On day 2, cells were washed with PBS and switched to medium B with (lanes 5–8) or without (lanes 1–4) 50 μM sodium compactin and 50 μM sodium mevalonate. In addition, cells were treated with 20 mM sodium mevalonate (lanes 2 and 6), 1 μM of the LXR-agonist T0901317 (lanes 3 and 7), or a combination of both (lanes 4 and 8). (A) After a 16-h incubation at 37°C, cells were harvested, and aliquots of total RNA (20 μg) from pooled dishes were hybridized for 10 min at 68°C to the indicated ³²P-labeled cRNA probes. RNase-protected fragments were separated by gel electrophoresis and exposed to film for 8 h at −80°C. (B) After a 16-h incubation at 37°C, cells received 25 μg/ml ALLN and were harvested 1 h later. Membranes and nuclear extract fractions were prepared, and aliquots (54 μg protein per lane) were subjected to SDS/PAGE. In A and B, immunoblot analysis was carried out with 5 μg/ml rabbit anti-rat SREBP-1 (Upper) or anti-rat SREBP-2 (Lower). Filters were exposed for 1 s at room temperature. P, SREBP precursor forms; N, nuclear cleaved forms of SREBP-1c and SREBP-2.