Journal: eLife

Article Title: Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

doi: 10.7554/eLife.02882

Figure Lengend Snippet: On day 0, SV-589 cells were set up in medium A at 1 × 10 5 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 ) 125 I-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 125 I-PFO * (11 × 10 3 cpm/μg). After 2 hr at 4°C, the total amount of cell surface binding of 125 I-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 125 I-PFO * binding resulting from SMase treatment. DOI: http://dx.doi.org/10.7554/eLife.02882.005

Article Snippet: Immediately afterward, 20 μl of a Ceramide/Sphingolipid Internal Standard Mixture II (Avanti Polar Lipids, Alabaster, AL) was added, and the mixtures were vortexed and sonicated at 40°C for 10 min. Lipids from the homogenate were extracted with 2 ml of 85:15 (vol/vol) ethyl acetate:isopropanol.

Techniques: Binding Assay, Incubation, Cell Culture, Purification

Journal: eLife

Article Title: Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

doi: 10.7554/eLife.02882

Figure Lengend Snippet: Major subspecies of sphingomyelin and ceramide in sterol-repleted (−compactin) and sterol-depleted (+compactin) PMs from SV-589 cells treated without and with SMase DOI: http://dx.doi.org/10.7554/eLife.02882.007

Article Snippet: Immediately afterward, 20 μl of a Ceramide/Sphingolipid Internal Standard Mixture II (Avanti Polar Lipids, Alabaster, AL) was added, and the mixtures were vortexed and sonicated at 40°C for 10 min. Lipids from the homogenate were extracted with 2 ml of 85:15 (vol/vol) ethyl acetate:isopropanol.

Techniques:

Journal: eLife

Article Title: Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

doi: 10.7554/eLife.02882

Figure Lengend Snippet: On day 0, SV-589 cells were set up in medium A at 1 × 10 5 cells per 60-mm dish. On day 3, cells were refed with medium B. On day 4, cells were treated for 1 hr at 37°C in medium B containing 50 µM mevalonate, 50 µM compactin, and 0–2% of HPCD as indicated for each point. The cells were then washed twice with PBS at room temperature, after which each monolayer received fresh medium B containing 50 µM compactin and 50 µM mevalonate in the absence or presence of 100 miliunits/ml of SMase. After incubation for 30 min at 37°C, one set of dishes was used for 125 I-PFO * binding and a parallel set was used for PMs purification. For 125 I-PFO* binding, 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 125 I-PFO * (10 × 10 3 cpm/µg) for 2 hr at 4°C. The total amount of cell surface 125 I-PFO * binding was determined. Each value represents the average of duplicate incubations. For PM purification, six 60-mm dishes for each HPCD treatment were pooled together, and the PMs were purified as described in ‘Materials and methods’. Lipids were extracted from the PM samples, and the content of unesterified cholesterol, phospholipids, and ceramide was measured. Each value represents the average of duplicate measurements of each pooled sample. The graph shows the amount of cell surface 125 I-PFO * binding plotted as a function of the cholesterol content of the PM. DOI: http://dx.doi.org/10.7554/eLife.02882.012

Article Snippet: Immediately afterward, 20 μl of a Ceramide/Sphingolipid Internal Standard Mixture II (Avanti Polar Lipids, Alabaster, AL) was added, and the mixtures were vortexed and sonicated at 40°C for 10 min. Lipids from the homogenate were extracted with 2 ml of 85:15 (vol/vol) ethyl acetate:isopropanol.

Techniques: Incubation, Binding Assay, Purification