LRAT-mediated LD formation in the absence of preexisting LDs. (A) Confocal microscopy of CHO-k1 cells expressing LRAT-GFP. After preincubation with medium containing 1% FBS, cells were incubated overnight with or without 200 µM OA or 20 µM ROH in the presence or absence of 1 µg/ml triacsin C. Cells were stained with DAPI (blue) and LD540 (green). Scale bars indicate 10 µm (zoomed regions are 10 µm wide). CON, control. (B) Quantification of predominant RE species by LC-MS/MS of WT and 4Δ yeast cells expressing LRAT 2 h after incubation in the presence or absence of 2 mM ROH. Amounts are expressed in pmol RE per μg protein. Bar plot indicates mean ± SD. (C) Widefield microscopy of Erg6-mCherry expressing WT and 4Δ yeast cells, with or without expressing LRAT, 2 h after incubation with or without 2 mM ROH (− or +). Images of UV autofluorescence (green), Erg6-mCherry (red), and brightfield were taken. Scale bars indicate 5 µm. (D) EM of WT and 4Δ yeast cells, with or without expressing LRAT, incubated with or without 2 mM ROH. M, mitochondria; N, nucleus. Scale bars indicate 250 nm. (E) EM of yft2Δscs3Δ and 4Δ yeast cells expressing LRAT incubated with 2 mM ROH. Scale bars indicate 100 nm. ER is indicated by yellow lines in right panels; LDs are indicated by red lines in right panels. (F) Widefield microscopy of Erg6-mCherry expressing WT yeast cells expressing LRAT. Two time series of UV autofluorescence (white), Erg6-mCherry (red), and brightfield taken 2, 4, 6, and 8 min after addition of 2 mM ROH. Arrows indicate LDs that become UV⁺ over time. Scale bars indicate 5 µm.