Role of sterols in vacuolar domain formation. (A) WT cells were grown in medium with FM4-64 to Stat-phase and visualized live by fluorescence microscopy. (B) Intact vacuoles from Stat-phase cells expressing Vph1-GFP were incubated with filipin for 30 min and imaged by fluorescence microscopy. Fluorescence intensities of filipin and GFP around the vacuole (arrows) were plotted. (C) 5 µM fenpropimorph or vehicle (DMSO) was added to growing cells expressing Vph1-GFP, and the cells were visualized after 16 h when they had reached Stat-phase. (D) Intact vacuoles from cells expressing Vph1-GFP were incubated with 10 mM MβCD for 30 min and imaged by fluorescence microscopy. Stat, Stat-phase; log, logarithmic growth phase. (E) Percentage of erg1Δ cells expressing Vph1-GFP that contained vacuoles with membrane domains when grown in YPD with ergosterol or cholesterol. The percentage of domains was calculated from 100–300 cells or vacuoles from two independent experiments. Error bars show means ± SD. Bars: (A, C, and D) 5 µm; (B) 1 µm.