Figure 7.

SREBPs regulate expression of insig-1 mRNA, but not insig-2 mRNA in hamster cells. (A) On day 0, N-BP cell lines were set up at 1 × 10⁶ cells per 100-mm dish in medium B. On day 2, the cells were switched to medium A supplemented with 5% lipoprotein-deficient serum and the indicated amount of ponasterone A. After incubation for 24 h at 37°C, the cells were harvested for preparation of total RNA. Total RNA (10 μg per lane) was subjected to electrophoresis and hybridized for 1 h at 68°C with ³²P-labeled probes against hamster insig-1, hamster insig-2, and human β-actin (1.5 × 10⁶ cpm/ml for each probe) as described in Supporting Materials and Methods. Filters were exposed to films with intensifying screens at −80°C for 2 days (insig-1), 7 days (insig-2), or 3 h (β-actin). (B) On day 0, cells were set up in medium B at the following density: 5 × 10⁵ cells per 100-mm dish (CHO-K1, CHO/pS2P, and SRD12B cells) and 8 × 10⁵ cells per 100-mm dish (M19 and SRD13A cells). On day 2, the cells were switched to medium C in the absence or presence of 1 μg/ml of 25-hydroxycholesterol plus 10 μg/ml of cholesterol (sterols). After incubation for 18 h at 37°C, the cells were harvested, and total RNA was analyzed as described in A. Filters were exposed to films at −80°C for 5 days (insig-1 and -2) and 1 h (β-actin).