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Fig. 6. Esterification of 7,7-azo[³H]cholestanol ([³H]AC) in intact 25RA and CT43 cells. Monolayers of 25RA and CT43 cells were incubated for 1-3 h as indicated at 37°C in medium D [Ham’s F-12 with 5% delipidated FBS (1), plus 35 mM oleic acid, and 10 mg/ml gentamycin] in the presence of 5 mM methyl-b-cyclodextrin (CD) complexed with [³H]AC in buffer (CD per sterol molar ratio = 64:1, with [³H]AC at 34.0 m Ci and 3.0 nmol). After incubation, the cells were washed with ice-chilled PBS three times. The cell extracts were harvested, the cellular lipids were extracted and analyzed by TLC, and the percentage esterification was determined as described (2).

Fig. 7. Effect of various agents on intact cell photolabeling of Niemann-Pick type C1 (NPC1)-YFP. (A) After incubation as monolayers in medium A in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 10 m g/ml U-18666A for 15 min, or for 36 h at 37°C as indicated, the CT60-NPC1-YFP cells (2.5 ´ 10⁶ cells) were detached, pelleted, and photolabeled in buffer with or without 10 m g/ml U-18666A in 1.0% ethanol (EtOH). U-18666A was from a 1.0 mg/ml stock solution in EtOH. Cells untreated with U-18666A received the same amount of EtOH only. Results are representative of two experiments. (B) Monolayers of CT60-NPC1-YFP cells grown in medium A were treated with bafilomycin A1 (0, 8, 66, or 535 nM as indicated) in 0.5% DMSO for 15 min at 37°C. The cells (2.5 × 10⁶ cells) were then detached, pelleted, and photolabeled according to procedures described in Materials and Methods, with bafilomycin A1 (0, 8, 66, or 535 nM as indicated) present in the incubation buffer. Bafilomycin A1 was from a 107-m M stock solution in DMSO. Cells untreated with bafilomycin A1 received the same amount of DMSO only. Results are representative of two experiments.

1. Chin, J. & Chang, T. Y. (1981) J. Biol. Chem. 256, 6304-6310.

2. Cruz, J., Thomas, M., Wong, E., Ohgami, N., Chang, C. C. Y., Curphey, T. & Chang, T. Y. (2002) J. Lipid Res. 43, 1341-1347.