Fsp27 promotes lipid exchange between contacted LDs. (A and B) Fsp27 promotes lipid exchange between contacted LDs. (A) LD images in live 3T3-L1 preadipocytes expressing Fsp27-GFP were captured manually. 1, 2, and 3 (a) represent LD1, LD2, and LD3, respectively. The line profile at the right side of each image represents the fluorescent intensity of neutral lipids (red) and Fsp27-GFP (green) along the white arrow line. The black arrowhead (d) marks the newly formed LDCS between LD2 and LD3. The box represents the photobleached area. (B) MOI percentage in the bleached (white circle) or unbleached (blue circle) region was obtained from four independent experiments. (C) Lipid exchange rates in 3T3-L1 preadipocytes expressing Fsp27-GFP (with or without GFP illumination), untagged Fsp27, and Cidea-GFP. NS, no significant difference. ***, P < 0.001. At least six FRAP analysis data for each group were used for calculation. (D) Western blotting showing levels of GFP, Fsp27-GFP, untagged-Fsp27, and Cidea-GFP in C. IB, immunoblotting. (E) Quantitative analysis from three independent experiments showing the activity of Fsp27-GFP and Cidea-GFP to enlarge LDs in 3T3-L1 preadipocyte. A large LD is defined as an LD with a diameter ≥2.5 µm. (F) Lipid exchange activity in various cell types. Yes or No represent Fsp27 that is or is not enriched at LDCSs, respectively. 0/20 represents no active lipid diffusion in 20 LD pairs/clusters. siFsp27 represents Fsp27 knockdown 3T3-L1 adipocytes. (G and H) Lipid exchange in MEF-derived wild-type (Fsp27^+/+; G) and Fsp27-deficient (Fsp27^−/−; H) adipocytes. Photobleaching was repeated for three times, and arrowheads point to the starting point of each bleaching. White circles represent bleached regions. (B, C, E, and H) Error bars represent SD. Bars: (A, G, and H) 5 µm; (B) 2 µm.