Fig. 4. The ApoB-crescent membrane is enriched with free cholesterol. (A) Huh7 cells treated with 10 µM ALLN for 12 hours were incubated with filipin (green) to label free cholesterol-enriched membrane. Filipin stained a crescent-shaped area around LDs (arrowheads). In most LDs, filipin staining was found on the same side as ApoB (red, goat polyclonal; the left panel), but on the opposite side from ADRP (red; the right panel). LDs were stained by BODIPY493/503 (blue). Bars, 5 µm. (B) The ALLN-treated Huh7 cells were treated with filipin and observed by EM. High-magnification images are shown in the right. Note that the membrane of the ApoB-crescent shows wavy deformation (arrowheads in the lower right figure), indicating the presence of filipin-cholesterol complexes. The membranes of mitochondria (Mt) and sER were smooth and not deformed (arrows in the upper right figure). Bar, 200 nm.