Fig. 8. Cholesterol is decreased in AP-3–/– fibroblasts. (A) The distribution of unesterified cholesterol is unaffected by the loss of AP-3. AP-3+/+ and AP-3–/– mouse fibroblasts were stained with filipin to detect unesterified cholesterol and were imaged by epifluorescence and deconvolution microscopy. Representative images are shown. (B) Total unesterified cholesterol levels in AP-3+/+ and AP-3–/– mouse fibroblasts were measured by flow cytometry of filipin-labeled cells. A representative histogram is shown with unlabeled and labeled AP-3–/– and AP-3+/+ cells in black and gray, respectively. (C) Quantification of filipin fluorescence intensities for AP-3+/+ and AP-3–/– fibroblasts normalized to unlabeled cells. Results are the average of three independent experiments each performed in triplicate. Standard deviations are indicated. (D) Biochemical quantification of total cellular cholesterol levels in AP-3+/+ and AP-3–/– fibroblasts was assessed using an Amplex Red Cholesterol Assay Kit. Fibroblasts were either untreated or treated with M
CD. Cholesterol content (µg cholesterol/µg protein for each genotype calculated as a percentage of AP-3+/+) for AP-3+/+ and AP-3–/– cells are shown. All determinations were performed at least in triplicate in three independent experiments (n=11). Standard deviations and statistically significant differences (P<0.002) are indicated.