Fig. 5. Cells expressing F508 CFTR show accumulation of glycosphingolipids as well as cholesterol, which is blocked in transfer to the Golgi and ER for esterification. (A) Transfer of NBD-cholesterol to the Golgi and ER is blocked in cells expressing F508 CFTR. BHK-21 cells were grown at 27°C for 48 hours, labeled with NBD-cholesterol and chased for 4 hours with growth medium at 37°C. The histogram shows that the percentage of cells with perinuclear Golgi staining is drastically reduced in cells expressing F508. At least 75 cells were counted and values are representative of three independent experiments. (B) Nile Red staining is decreased in cells expressing F508 CFTR. Nile Red staining of BHK-21 cells grown at 27°C was reduced in cells expressing F508 CFTR. The average Nile Red intensity per cell is shown in a histogram. At least 90 cells were counted from three different experiments and standard deviations are indicated. (C) Distribution of fluorescent sphingolipid analog is altered in cells expressing F508 CFTR. BHK-21 cells expressing CFTR or F508 at 27°C for 48 hours were labeled with BODIPY-LacCer for 30 minutes at 4°C and subsequently chased for 1 hour at 37°C. The histogram shows that the percentage of cells with Golgi staining is reduced in cells expressing F508 CFTR.