LDs containing REs have a distinct morphology. (A) Chemical structures of the neutral lipids RP, cholesteryl oleate, and TOG. (B) Reaction mechanism of LRAT. (C and D) Confocal microscopy of murine HSCs (mHSCs) cultured for 1, 3, 6, or 8 d and stained with DAPI (blue), LD540 (green), and anti-desmin (red, an HSC marker; C), or DAPI (blue) and anti–α-smooth muscle actin (red, a marker of activated HCSs; D). Scale bars indicate 50 µm. Zoomed regions are 16.4 µm wide. (E) Relative expression of LRAT mRNA in mHSCs after 1 or 7 d of culture by quantitative PCR. Bar plot indicates mean ± SEM of three animals. Statistical significance was determined by two-tailed paired Welch’s t test. (F) Confocal microscopy of mHSCs 2 h after isolation showing UV autofluorescence (blue), LD540 (green), and anti-desmin (red). Bottom panel is a zoomed inset of the area surrounded by dotted lines in the main panel. Closed triangles indicate UV^─ LDs; open triangles indicate UV⁺ LDs. Scale bars indicate 50 µm. Zoomed regions are 14.5 µm wide. (G–I) Confocal microscopy of CHO-k1 cells expressing GFP or LRAT-GFP incubated overnight in the presence or absence of 20 µM ROH or 200 µM OA. Imaged channels are DAPI (blue) and LD540 (green; G) or UV autofluorescence (left) and LD540 (middle; I). Scale bars indicate 10 µm. Zoomed regions are 5 µm wide. CON, control. (H) Quantification of G showing LD size (left panel), LD number per cell (middle panel), and cumulative LD volume by LD size (right panel). Conditions are colored in black (control), red (200 µM OA), and blue (20 µM ROH; in the respective conditions, LDs counted were n = 1,436, 4,064, 1,127, 1,095, 7,373, and 1,132, respectively; cells counted were n = 55, 28, 65, 47, 93, and 82, respectively). (J) Full projections of 3D-SIM images of CHO-k1 cells expressing LRAT-GFP incubated overnight in the presence 20 µM ROH or 200 µM OA. Bottom panels are zoomed insets (10.8 µm wide) of areas surrounded by dotted lines in the top panels. Scale bars indicate 10 µm. **, P < 0.01; ***, P < 0.001.