Dynamics of lipid incorporation into pre- and emerging LDs. (A) Starved OFP-HPos–transfected cells (top) were treated with OA and FA-BODIPY (bottom) and followed after 15 min by video microscopy (Video 2). Blue and green arrows indicate FA-BODIPY accumulation in pre-LDs. White circles indicate pre-LDs, and red circles emerging LDs. Arrows indicate a representative pre-LD. The degree of colocalization between HPos and FA-BODIPY at different times is indicated by the color of the arrow; red denotes a lack of colocalization, blue partial colocalization, and green complete colocalization. (B) A high-magnification sequence of lipid incorporation into the pre-LD indicated with a red arrow in the first panel of A (Video 3). The arrows mark the pre-LD selected in A. (C) A high-magnification sequence of lipid incorporation into the emerging LD indicated with a blue arrow in the last panel of A (Video 3). Arrows mark a representative emerging LD. (D) Cells were treated as in A, and the FA-BODIPY intensity in at least 30 pre-LDs (black circles), emerging LDs (red circles), and the cytosol (gray circles) was quantified before and after OA in three independent experiments. Error bars indicate the standard deviation between experiments. ***, P < 0.001. (E) Thin-layer chromatography of fluorescent lipids in cells incubated 30, 60, or 120 min with OA and FA-BODIPY or 120 min but in the presence of 10 µM Triacsin C (TC). Bars: (A) 2 µm; (B and C) 0.5 µm.