(A) Deuterated water (D2O) labeling of lanosterol in cultured cells. (Top) Isotopomer spectrum of lanosterol in SV-589 cells grown for 24 hr in the absence (left) or presence (right) of 5% D2O added to the medium. (Bottom) Turnover of lanosterol in SV-589 cells. Cells were grown in NCLPPS (open circles) or in NCLPPS plus 25-hydroxycholesterol (1 μg/ml) (closed circles) to ∼60% confluence. After 16 hr , the medium was supplemented with 5% D2O and cells were harvested at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hr (last two points not shown, but used for modeling). Sterols were analyzed by LC-MS/MS and the fraction of lanosterol that was newly synthesized was determined using isotopomer analysis (IA) (see Figure 2—figure supplement 1) and the results were fit to a first-order kinetic model (solid lines) as described in the ‘Materials and methods’. The rates of synthesis of lanosterol in the presence or absence of sterols were calculated by multiplying the first-order rate constant by the concentration of lanosterol. Standard deviations are reported based on four independent replicates. (B) Biosynthetic rates of intermediary sterols in cultured cells. The rate of synthesis of intermediary sterols in the Bloch (left) and K–R (right) pathways was measured using D2O labeling. Cells were plated at a density of 500,000/60 mm dish and grown to ∼60% confluence. D2O was then added to the medium to a final concentration of 5% (vol/vol). Cells were harvested at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hr, and lipids were extracted using methanol-dichloromethane. Sterols were analyzed by LC-MS/MS and the fraction of each sterol that was newly synthesized was determined using IA and the results were fit to a first-order kinetic model as described in the ‘Materials and methods’. Y1-BS1 cells, a mouse adrenal cell line (Top) were grown in 15%horseserum. SV-589 cells, an immortalized human skin fibroblast line (bottom), were grown in either 10% FCS. Means and standard deviations are reported based on four independent replicate experiments in each cell line. ***p < 0.001.