Figure 3. Effect of SMase treatment of human fibroblasts on amount of cell surface binding of 125I-PFO* (A) and PM content of SM and ceramide (B–D).
On day 0, SV-589 cells were set up in medium A at 1 × 105 cells per 60-mm dish (A–D). On day 2, cells were switched to lipoprotein-deficient medium C. On day 3, cells were treated with fresh medium B containing 50 μM sodium mevalonate in the presence or absence of 50 μM compactin as indicated. On day 4, each monolayer received fresh medium B containing 50 μM mevalonate in the absence or presence of 50 μM compactin and 100 milliunits/ml of SMase as indicated. (A) 125I-PFO* binding. After incubation for 15 min at 37°C, the cells were washed five times as described in ‘Materials and methods’ and then incubated with 2 ml of ice-cold buffer A containing 25 μg/ml 125I-PFO* (11 × 103 cpm/μg). After 2 hr at 4°C, the total amount of cell surface binding of 125I-PFO* was determined. Each bar represents the mean of triplicate incubations with individual values shown. (B–D) Lipid measurements. Cells were cultured under identical condition as described above. For each treatment, six 60-mm dishes were pooled together for purification of PMs by surface biotinylation as described in ‘Materials and methods’. Lipids were extracted from the membranes, and the content of cholesterol (B), SM (C and D), and ceramide (C and D) were measured as described in ‘Materials and methods’. The data represent the mean ± SEM obtained from three independent experiments. Each individual data point denotes the average of duplicate measurements of each pooled sample. Bracketed numbers denote the increase in 125I-PFO* binding resulting from SMase treatment.
Figure 3—figure supplement 1. Movement of FCS-derived cholesterol and effect of SMase treatment in hamster cells.
) or 0.2 mM sodium [14C]oleate-albumin (4466 dpm/nmol) (
), the cells were harvested for assays. For 125IPFO* binding (), after the indicated time the cells were washed five times as described in ‘Materials and methods’ and then incubated with 2 ml ice-cold buffer A containing 25 μg/ml 125IPFO* (45 × 103 cpm/μg). After 2 hr at 4°C, the total cell surface binding of 125I-PFO* was determined, and the amount bound after subtraction of the zero-time value (0.4 μg/mg protein) is plotted as ‘Increase in 125I-PFO* Bound’. For measurement of cholesteryl [14C]oleate formation (), after the indicated time the cells were harvested, and the increase in content of cholesteryl [14C]oleate was determined after subtraction of the zero-time value (0.0 nmol/mg protein). All values shown are the average of duplicate incubations. (B) Effect of SMase treatment of hamster cells on amount of cell surface binding of 125I-PFO*. On day 0, CHO-K1 cells were set up in medium F at 4 × 105 cells per 60-mm dish. On day 1, cells were switched to lipoprotein-deficient medium G. On day 2, cells were treated with fresh medium G containing 50 μM sodium mevalonate in the presence or absence of 10 μM compactin as indicated. On day 3, each monolayer received fresh medium G containing 50 μM mevalonate in the absence or presence of 10 μM compactin and 100 milliunits/ml of SMase as indicated. After incubation for 30 min at 37°C, the cells were washed five times as described in ‘Materials and methods’ and then incubated with 2 ml of ice-cold buffer A containing 25 μg/ml 125I-PFO* (10.5 × 103 cpm/μg). After 2 hr at 4°C, the total amount of cell surface binding of 125I-PFO* was determined. Each bar represents the average of duplicate incubations with individual values shown. Bracketed numbers denote the increase in 125I-PFO* binding resulting from SMase treatment.